KR101871128B1 - Combination alzheimer therapy using anti-n3pglu abeta antibodies + a bace inhibitor - Google Patents

Abstract

The invention encompasses administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody to a patient in need of treatment of a cognitive or neurodegenerative disease , ≪ / RTI > cognitive or neurodegenerative diseases.
(I)

Description

COMBINATION ALZHEIMER THERAPY USING ANTI-N3PGLU ABETA ANTIBODIES + A BACE INHIBITOR USING ANTI-N3pGlu A BETA ANTIBODY +

The present invention relates to a combination of a BACE inhibitor and an anti-N3pGlu A beta monoclonal antibody, and a method of using the same to treat certain neurological disorders such as Alzheimer's disease.

The present invention is in the field of the treatment of Alzheimer's disease and other diseases and disorders involving amyloid beta (A beta) peptides which are neurotoxic and highly aggregative peptide fragments of amyloid precursor protein (APP). Alzheimer's disease is a devastating neurodegenerative disorder affecting millions of patients worldwide. There is a substantial unmet need in the treatment of Alzheimer ' s disease, in terms of currently marketed approved agents that provide only temporary, symptomatic benefits to the patient.

Alzheimer's disease is characterized by the formation, aggregation and deposition of A beta in the brain. Complete or partial inhibition of beta-secretase (beta-site amyloid precursor protein-cleaving enzyme; BACE) has been shown to have a significant effect on plaque-related and plaque-dependent pathology conditions in mouse models. This even produces a prolonged significant reduction of plaque burden and synaptic deficiency, even with a small decrease in the level of A beta peptide, thus providing a significant therapeutic benefit, particularly in the treatment of Alzheimer ' s disease.

Furthermore, antibodies that specifically target N3pGlu A beta have been shown to reduce in vivo plaque levels (US 2013/0142806). N3pGlu A beta, referred to also as N3pE or A beta _{-42 p3} is just distal of the A beta peptide is only found in the plaque-cut form. Although the N3pGlu A beta peptide is a secondary component of the deposited beta beta in the brain, the research has demonstrated that the N3pGlu A beta peptide has aggressive aggregation properties and accumulates early in the deposition cascade.

Combinations of BACE inhibitors and antibodies that bind to the N3pGlu A beta peptide are intended to provide treatment for A beta peptide-mediated disorders, such as Alzheimer ' s disease, which may be more effective than either drug alone. For example, treatment with such a combination may allow the use of lower doses of the drug (s) of either or both, as compared to each drug used alone, leading to lower side effects while potentially retaining efficacy . Aiming to eliminate the deposited form of A beta with N3pG antibody and BACE inhibitor can reduce or prevent further deposition of A beta by facilitating the phagocytosis of existing plaque deposits while at the same time inhibiting the formation of A beta will be.

US 2009/0209755 discloses fused amino dihydrothiazine derivatives that have BACE inhibitory activity and are further disclosed as useful therapeutics for neurodegenerative diseases caused by A [beta] peptides, such as Alzheimer type dementia. Also, (S) -4- (2,4-difluoro-5-pyrimidin-5-yl-quinazolin- Phenyl) -4-methyl-5,6-dihydro-4H- [l, 3] thiazin-2-ylamine. U.S. Patent No. 8,278,334 discloses a method of treating a cognitive or neurodegenerative disease, comprising administering a substituted cyclic amine BACE-1 inhibitor together with an anti-amyloid antibody. In addition, Neuroscience, 34 (35), pages 11621-11630 (2014), discloses that combination therapy using BACE inhibitors and anti-A beta- antibody gentenumap improves amyloid loss in APP _London mice.

Accordingly, the present invention provides a method of treating a cognitive or neurodegenerative disease, comprising administering to a patient in need thereof a therapeutically effective amount of a BACE inhibitor in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody. . ≪ / RTI >

More particularly, the invention relates to a method of treating a cognitive or neurodegenerative disease, comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody Or a neurodegenerative disease, comprising administering a therapeutically effective amount of a compound of the present invention.

(I)

Wherein R is H or F;

A is

이다.

to be.

The present invention also provides a method of treating a disease characterized by the formation and deposition of A beta comprising administering to a patient in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention further provides a method of treating Alzheimer's disease comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody, Provides a method of treating the disease.

The invention also relates to a method of treating mild Alzheimer's disease comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody, Thereby providing a method for treating Alzheimer's disease.

The present invention further provides a method of treating a mild cognitive impairment comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody. A method of treating a mild cognitive disorder.

The present invention further relates to a method for the treatment of Alzheimer's disease, comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody. Provides a method of treating bulb Alzheimer ' s disease.

The invention also provides a method of treating a mild cognitive impairment in a patient in need thereof, comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody A method of preventing progression of a mild cognitive impairment to Alzheimer ' s disease.

The present invention further relates to a method for the treatment of brain amyloid angiopathy (CAA) comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody (&Quot; CAA "). ≪ / RTI >

In addition, the invention relates to a pharmaceutical composition for the treatment of Alzheimer's disease, mild Alzheimer's disease, progenitor Alzheimer's disease, or for the prevention of the progression of mild cognitive impairment to Alzheimer's disease, for use in therapy, in particular an effective amount of an anti-N3pGlu A beta monoclonal Lt; RTI ID = 0.0 > I < / RTI >

The present invention further relates to a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof in combination with a pharmaceutical composition comprising an anti-N3pGlu A beta monoclonal antibody and at least one pharmaceutically acceptable carrier, diluent or excipient, Together with a pharmaceutically acceptable carrier, diluent or excipient.

The invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and an anti-N3pGlu A beta monoclonal antibody. The present invention further provides pharmaceutical compositions comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients, and a pharmaceutical composition comprising an anti-N3pGlu A beta monoclonal antibody, Together with a pharmaceutically acceptable carrier, diluent or excipient. As used herein, the term "kit" refers to a compound of formula I or a pharmaceutically acceptable salt thereof, wherein one component is a compound of formula I and the other component is a separate container of each component that is an anti-N3pGlu A beta monoclonal antibody . A "kit" is also intended to include a separate container of each of the components, one component being a compound of formula I or a pharmaceutically acceptable salt thereof, and the other component being a anti-N3pGlu A beta monoclonal antibody, Together with instructions for administration as a combination.

The present invention further relates to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, and an effective amount of a compound of formula I, for the manufacture of a medicament for the treatment of Alzheimer's disease, mild Alzheimer's disease, prodromal Alzheimer's disease or prevention of progression of mild cognitive impairment to Alzheimer's disease RTI ID = 0.0 > anti-N3 < / RTI > pGlu A beta monoclonal antibody.

One of ordinary skill in the relevant art is described in U.S. Patent No. 8,679,498 B2 (USSN 13 / 810,895) entitled " Anti-N3pGlu Amyloid Beta Peptide Antibody and Its Use "issued Mar. 25, Will recognize and recognize that "anti-N3pGlu A beta monoclonal antibody", and specific antibodies, "B12L" and "R17L", are identified and disclosed with the method of making and using the antibody. See, for example, Table 1 of U.S. Patent No. 8,679,498 B2.

In addition, the amino acid sequences for the specific antibodies used in the present invention are provided in Table A below:

Table A- Antibody Sequence Identification Number (SEQ ID NO)

Cognitive or neurodegenerative diseases include Alzheimer's disease, mild Alzheimer's disease, mild cognitive impairment, global Alzheimer's disease, brain amyloid angiopathy (CAA), Down's syndrome and the like.

The term "treating", "treating" or "treatment" as used herein refers to inhibiting, slowing, stopping, reducing or reversing the progression or severity of an existing condition, disorder, condition or disease.

The term "patient" as used herein refers to a human.

The term "inhibition of the production of A beta peptide" is considered to mean reducing the in vivo levels of A beta peptide in a patient.

The term "effective amount ", as used herein, refers to an amount or amount of a compound of formula I or a pharmaceutically acceptable salt thereof that provides a desired effect in a patient under diagnosis or treatment, Refers to the amount or dose of N3pGlu A beta monoclonal antibody. Combination therapy is provided by administering a compound of formula I or a pharmaceutically acceptable salt thereof of the invention together with an anti-N3pGlu A beta monoclonal antibody in a body to provide an effective level of a compound of formula I and an anti-N3pGlu A beta monoclonal antibody ≪ / RTI >

An effective amount can be readily determined by the use of known techniques and by a person skilled in the art as a matter of routine skill in the art by observing results obtained under similar circumstances. In determining an effective amount for a patient, the species of the patient; His size, age and overall health; The specific disease or disorder that accompanies it; The severity of the disease or disorder; Individual patient response; The particular compound administered; Mode of administration; Bioavailability characteristics of the agent to be administered; Selected dosing regimen; The use of concomitant medicines; And other relevant circumstances, are contemplated by the attending clinician.

The compounds of formula I and their pharmaceutically acceptable salts are effective in the combination of the present invention over a generally wide dosage range. For example, a daily dosage is generally from about 0.1 mg / day to about 1000 mg / day, preferably from about 0.1 mg / day to about 500 mg / day, and most preferably from about 0.1 mg / day to about 100 mg / day. In addition, anti-N3pGlu A beta monoclonal antibodies are generally effective over a wide dosage range in the combinations of the present invention. For example, a weekly dose is typically about 0.1 to 10 mg / kg / week, preferably about 0.3 to about 6 mg / kg / week, and most preferably about 0.3 mg / kg / week to about 3 mg / kg / week. In some cases, dosage levels below the lower limit of the above range may be greater than the appropriate amount, while in other cases larger doses may be used with acceptable side effects, And is not intended to limit the scope of the invention.

The BACE inhibitors and antibodies of the present invention are preferably formulated as a pharmaceutical composition to be administered by any route that makes the compound bioavailable. The route of administration may be varied in any way limited by the physical characteristics of the drug and the convenience of the patient and the caregiver. Preferably, the anti-N3pGlu A beta monoclonal antibody composition is for parenteral administration, such as intravenous or subcutaneous administration. In addition, a BACE inhibitor, such as a compound of Formula I or a pharmaceutically acceptable salt thereof, is for oral, parenteral or transdermal administration, including intravenous or subcutaneous administration. Such pharmaceutical compositions and methods for their preparation are well known in the relevant art. (See, for example, Remington: The Science and Practice of Pharmacy (D. B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).

As used herein, the phrase " in combination "means that a BACE inhibitor, such as a compound of formula I, or a pharmaceutically acceptable salt thereof, is co-administered with an anti-N3pGlu A beta monoclonal antibody, such as an anti-N3pGlu A beta monoclonal antibody, In any order, or any combination thereof. The two molecules may be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. BACE inhibitors may be administered prior to, concurrently with, or subsequent to, or in some combination with, an anti-N3pGlu A beta monoclonal antibody. When the anti-N3pGlu A beta monoclonal antibody is administered at repeated intervals (e. G. During standard procedures of treatment), the BACE inhibitor may be administered prior to each administration of the anti-N3pGlu A beta monoclonal antibody, , Or at a different interval with respect to therapy using the anti-N3pGlu A beta monoclonal antibody, or subsequent to or in some combination thereof, or prior to the treatment with the anti-N3pGlu A beta monoclonal antibody, May be administered at any time during, or subsequent to, single or sequential dose (s).

The following paragraphs describe preferred groups, substituents, and configurations of the present invention.

Preferred compounds are:

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide; And

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -3,5-difluoro-pyridine-2-carboxamide; And pharmaceutically acceptable salts thereof.

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide or a pharmaceutically acceptable salt thereof is particularly preferred.

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2- carboxamide is most preferred.

In addition, the crystalline form 2 of N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidin- , 4-d] [l, 3] thiazin-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide is the preferred compound; Characterized by a substantial peak in the X-ray diffraction spectrum at a diffraction angle 2-theta of 11.8 DEG with one or more peaks selected from the group consisting of 18.6 DEG, 19.3 DEG and 26.7 DEG with a tolerance for diffraction angle of 0.2 DEG Crystalline form 2 < RTI ID = 0.0 > N- [3 - [(4aR, 7aS) -2-Amino- 6- (5-fluoropyrimidin- 2- yl) -4a, 5,6,7-tetrahydropyrrolo [ -d] [l, 3] thiazin-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2- carboxamide is particularly preferred.

Preferred anti-N3pGlu A beta monoclonal antibodies are B12L and R17L identified in U.S. Patent No. 8,679,498 B2 (see, e. G., Table 1 thereof), with B12L being particularly preferred.

One of ordinary skill in the relevant art will recognize that compounds of formula I can exist in tautomeric forms as shown in Scheme A. In the present application, given any reference to one of the specific tautomers of a compound of formula I, it is understood that it encompasses both tautomeric forms and all mixtures thereof.

<Reaction Scheme A>

For clarity, no specific stereochemistry center has been specified in the following reaction scheme and certain substituents have been removed, which is not intended to limit the teaching of the reaction schemes in any way. In addition, individual isomers, enantiomers, and diastereoisomers can be separated or resolved by conventional techniques in the art at any convenient point in the synthesis of compounds of formula I by methods such as selective crystallization techniques or chiral chromatography (See, for example, J. Jacques et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, Inc., 1981, and EL Eliel and SH Wilen "Stereochemistry of Organic Compounds ", Wiley -Interscience, 1994). The terms "isomer 1" and "isomer 2" refer to the first and second eluting compounds, respectively, from chiral chromatography and when chiral chromatography is initially disclosed in the synthesis, the same names apply to subsequent intermediates and examples do.

Those of ordinary skill in the relevant art will recognize that the compounds of the present invention are composed of cores containing at least two chiral centers.

<Reaction Scheme B>

Although the present invention encompasses all individual enantiomers of the compounds as well as mixtures of enantiomers, such as racemates, compounds in which the absolute configuration at the carbon labeled 1 and 2 as illustrated in Scheme B is the preferred / RTI &gt;

Additionally, certain intermediates described in the following schemes may contain one or more nitrogen protecting groups. The variable protecting groups may in each case be the same or different depending on the particular reaction conditions to be carried out and the particular conversion. Protecting and deprotecting conditions are well known to those of ordinary skill in the art and are described in the literature (see, for example, Greene's Protective Groups in Organic Synthesis, Fourth Edition, by Peter GM Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

The compounds of formula I, or pharmaceutically acceptable salts thereof, may be prepared by a variety of procedures known in the art, some of which are illustrated in the following preparations and examples. The specific synthetic steps for each route described may be combined in different ways or combined with the steps from the different schemes to produce a compound of formula I or a salt thereof. The products of each step in the following reaction schemes can be recovered by conventional methods well known in the relevant art including extraction, evaporation, precipitation, chromatography, filtration, softening and crystallization. Also, all substituents are as defined above, unless otherwise indicated. The reagents and starting materials are readily available to those of ordinary skill in the relevant art.

As used herein, "APP" refers to an amyloid precursor protein; "BOC" refers to tert-butoxycarbonyl; "BSA" refers to bovine serum albumin; "CSF" refers to cerebrospinal fluid; "DCC" refers to 1,3-dicyclohexylcarbodiimide; "DIC" refers to diisopropylcarbodiimide; "DCM" refers to dichloromethane; "DIPEA" refers to diisopropylethylamine; "DMAP" refers to dimethylaminopyridine; "DMEM" refers to Dulbecco ' s Modified Eagle ' s Medium; "DMSO" refers to dimethyl sulfoxide; "EDCI" refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; "EDTA" refers to ethylenediamine tetraacetic acid; "ee" refers to the enantiomeric excess; "EtOAc" refers to ethyl acetate; "Ex" refers to an embodiment; "F12" refers to Ham (F12) medium; "FBS" refers to fetal bovine serum; "FRET" refers to fluorescence resonance energy transfer; "HATU" refers to (dimethylamino) -N, N-dimethyl (3H- [1,2,3] triazolo [4,5- b] pyridin- 3- yloxy) methaniminium hexafluorophosphate ; "HEK" refers to human embryonic kidney; "hr" refers to time; "HOAc" refers to acetic acid; "HOAt" refers to 1-hydroxy-7-azobenzotriazole; "HOBt" refers to 1-hydroxylbenzotriazole hydrate; "HBTU" refers to 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; "HRP" refers to horseradish peroxidase; "IC ₅₀ " refers to the concentration of agonist that produces 50% of the maximal inhibition response possible for an agonist; "iPr" refers to isopropyl; "min" refers to minutes; "MTBE" refers to methyl tert-butyl ether; "PBS" refers to phosphate buffered saline; "PDAPP" refers to a platelet derived amyloid precursor protein; "Prep" refers to manufacture; "psi" refers to pounds per square inch; "PyBOP" refers to benzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate; "PyBrop" refers to bromo-tris-pyrrolidinophosphonium hexafluorophosphate; "RFU" refers to the relative fluorescence unit; "Rt" refers to the residence time; "SCX" refers to strong cation exchange chromatography; "SFC" refers to supercritical fluid chromatography; "SEM" refers to the standard error of the mean; "THF" refers to tetrahydrofuran and "TMB" refers to 3,3 ', 5,5'-tetramethylbenzidine.

The following preparations and examples further illustrate the present invention.

In the following schemes, all substituents are as defined above unless otherwise indicated. Reagents and starting materials are generally readily available to those of ordinary skill in the relevant art. Others may be prepared by the procedures described in the following Preparations and Examples, including the standard techniques of organic and heterocyclic chemistry, and any novel procedures.

<Reaction Scheme 1>

Scheme 1 illustrates the formation of the oxime (the product of Step 2) and (the product of Step 5). Oximes can each be used to form bicyclic isoxazole (the product of step 3 or 7). Substituted aromatic groups can be inserted at different points in the synthesis as shown in Scheme 1, Step 1 and Step 7. "PG" is a protecting group generated for an amino group such as carbamate and allyl. Such groups are well known and recognized in the relevant art.

In a two-step reaction, the ketone with a beta halogen is alkylated with an allyl amine protected with an inorganic base, such as potassium carbonate (step 1), followed by reaction with a hydroxylamine hydrochloride and an organic base such as, for example, Treatment with pyridine can afford the oxime (step 2). The oxime product of step 2 is then converted to the bicyclic isoxazole in the 3 + 2 cyclization by several methods such as heating the oxime of step 2 in a non-polar solvent such as toluene or xylene to form bicyclic isoxazole (Step 3). Alternatively, the oxime can be formed starting from dimethylacetal, which is treated with an acid such as formic acid to form the aldehyde (the product of Step 4). In step 5, the aldehyde of step 4 can be converted to the oxime product of step 5 using hydroxylamine hydrochloride and a base such as sodium acetate trihydrate. The bicyclic isoxazole product of Step 6 can be formed from the oxime as shown in Step 6 using an aqueous solution of sodium hypochlorite. In step 7, the protected bicyclic isoxazole is then reacted with an aromatic organolithium reagent or a Grignard reagent to yield the protected bicyclic isoxazole product of step 7.

<Reaction Scheme 2>

Scheme 2 illustrates the different pathways to the protected pyrrolothiazine product of step 10 or step 12. [ Protected bicyclic isoxazole can be treated with Raney Nickel in powdered Zn in acetic acid or in a polar solvent such as ethanol under pressure hydrogenation conditions to afford aminopyrrolidine methanol (the product of Step 11) . Subsequently, the aminopyrrolidine methanol product of Step 11 is reacted with benzoyl isothiocyanate in a polar solvent such as THF, then 1,1-carbonyldiimidazole (CDI) is added to form the fused protected pyrrolidine thiazine (The product of Step 12). Alternatively, the amine of bicyclic isoxazole is reacted with benzoyl isothiocyanate to provide thiourea (the product of step 8), followed by ring opening of the isoxazole ring in powdered zinc in acetic acid in step 9 to give the product of step 9 Of the hydroxyl compound product. The hydroxyl compound is then treated with 1-chloro-N, N, 2-trimethylpropenylamine in a polar aprotic solvent such as CDI or DCM in THF to form the fused protected pyrrolidine thiazine (the product of Step 10) Can be formed. Fused pyrrolidine thiazines can also be formed from Mitsunobu reactions such as using triphenylphosphine and diisopropyl azodicarboxylate (DIAD).

<Reaction Scheme 3>

Scheme 3 shows the conversion of the pyrrolothiazines to aniline (the product of Step 13), which can then be acylated followed by deprotection and heteroarylation of the pyrrolidine. Acylation of aniline nitrogen and deprotection of thiazine amine leads to compounds of formula I.

The azido-dehalogenation is carried out on an appropriate pyrrolothiazine in the presence of an azide source, such as sodium azide. Such azido-dehalogenation reactions are well known and recognized in the relevant art. Reduction of the resulting azide intermediate to aniline (the product of Step 13) can be carried out by hydrogenation conditions which are well known and described in the relevant art, or by reducing agents such as LiAlH ₄ , NaBH ₄ , and PPh ₃ &lt; / RTI &gt;

The BOC protected pyrrolidine can be deprotected under acidic conditions well known in the relevant art (Step 1 of Step 14). Subsequently, the deprotected pyrrolidine is reacted with a heteroaryl (heteroaryl) heteroaryl group by nucleophilic aromatic substitution (SNAr) with an organic base such as dipea, triethylamine or a substituted aromatic pyrimidine using N, N, To give the product of Step 2 in Step 14. The aniline product of Step 14 can be coupled with a heteroaromatic carboxylic acid under coupling conditions (the product of Step 1, Step 15). One of ordinary skill in the relevant art will recognize that there are a number of methods and reagents for the formation of amides resulting from the reaction of carboxylic acids and amines. For example, reaction of an appropriate aniline with an appropriate acid in the presence of a coupling reagent and an amine base such as DIPEA or triethylamine will provide the compound of formula I after deprotection of the thiazine amine. Coupling reagents include carbodiimides such as DCC, DIC, EDCI, and aromatic oximes such as HOBt and HOAt. Additionally, uranium or phosphonium salts of non-nucleophilic anions such as HBTU, HATU, PyBOP and PyBrOP may be used instead of more traditional coupling reagents. An additive such as DMAP can be used to enhance the reaction. Alternatively, protected aniline amines can be acylated using substituted benzoyl chlorides in the presence of a base such as triethylamine or pyridine. The protected thiazine amine can then be deprotected with a polar aprotic solvent such as an organic base such as pyridine and methylhydroxyamine hydrochloride in methanol or an inorganic base such as lithium hydroxide in methanol to give the compound of formula I.

In an optional step, a pharmaceutically acceptable salt of a compound of formula I may be formed by reaction of the appropriate free base of formula I with an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions. Additionally, the formation of such salts can occur simultaneously during deprotection of the nitrogen protecting group. The formation of such salts is well known and recognized in the relevant art. For example, Gould, P. L., "Salt selection for basic drugs," International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R. J., et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4: 427-435 (2000); And Berge, S. M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977). One of ordinary skill in the art can readily convert a compound of formula I into a pharmaceutically acceptable salt, such as a hydrochloride salt, and isolate it as a pharmaceutically acceptable salt, such as a hydrochloride salt.

Production Examples and Examples

The following preparations and examples further illustrate the present invention.

Production Example 1

1- (3-bromophenyl) -2- (diallylamino) ethanone

Potassium carbonate (38.8 g, 281 mmol) was added to 3-bromophenacyl bromide (60 g, 216 mmol) in acetonitrile (430 mL) and the mixture was cooled to 0 <0> C under nitrogen. Diallylamine (34.6 mL, 280.63 mmol) was added dropwise over 1 hour and the reaction allowed to warm to 22 &lt; 0 &gt; C overnight. The crude reaction mixture was concentrated and the residue was partitioned between water (300 mL) and MTBE (300 mL). The aqueous layer was discarded and the organic layer was washed with water (100 mL, 2 x) and brine (100 mL). The organic layer was dried over sodium sulfate, filtered and the solvent evaporated to constant weight to give the title compound (62 g, 98%). ES / MS (m / e): 294 (M + 1).

Production Example 2

Benzyl N- (2,2-dimethoxyethyl) carbamate

A solution of aminoacetaldehyde dimethylacetal (25 mL, 229 mmol) in toluene (120 mL) was treated at 0 C with 4.85 M sodium hydroxide solution (70.8 mL, 343.5 mmol). The mixture was stirred at 0 &lt; 0 &gt; C for 10 min and added while keeping the internal temperature below 20 &lt; 0 &gt; C while adding benzyl chloroformate (33.8 mL, 229 mmol). The mixture was allowed to warm to room temperature over 4 hours. The organic layer was separated, washed with brine, dried over sodium sulfate and concentrated to dryness to give the title compound (54 g, 98%). ES / MS (m / e): 240 (M + H).

Production Example 3

Benzyl N-allyl-N- (2,2-dimethoxyethyl) carbamate

A solution of benzyl N- (2,2-dimethoxyethyl) carbamate (50 g, 208.9 mmol) in toluene (180 mL) was treated with solid potassium hydroxide (51.6 g, 919.69 mmol) under nitrogen. After 10 minutes, benzyltriethylammonium chloride (0.8 g, 3.1 mmol) was added. After an additional 10 minutes, a solution of allyl bromide (33 g, 272.8 mmol) in toluene (50 mL) was added dropwise over 10 minutes. The resulting mixture was stirred at 50 &lt; 0 &gt; C for 48 hours. The mixture was cooled to room temperature and quenched with water. The organic layer was separated, washed with brine, dried over magnesium sulphate and concentrated to dryness to give the title compound (44 g, 75%). ES / MS (m / e): 280 (M + H).

Production Example 4

Benzyl N-allyl-N- (2-oxoethyl) carbamate

A solution of benzyl N-allyl-N- (2,2-dimethoxyethyl) carbamate (30 g, 107 mmol) in formic acid (36.8 mL, 860 mmol) and water (4.84 mL) was stirred overnight at room temperature. The mixture was concentrated and diluted with hexane / EtOAc (1: 2) and water. The organic layer was separated, washed with brine solution to pH = 6, and dried over sodium sulfate. Evaporation of the solvent gave the title compound (25 g, 99%). ES / MS (m / e): 234 (M + H).

Production Example 5

1- (3-bromophenyl) -2- (diallylamino) ethanone oxime

A solution of 1- (3-bromophenyl) -2- (diallylamino) ethanone (60 g, 204.7 mmol) in ethanol (720 mL) and pyridine (24.8 mL, 307 mmol) Lt; / RTI &gt; Hydroxylamine hydrochloride (17 g, 246 mmol) was added in portions to the solution over 1 h. The reaction was allowed to warm to 50 &lt; 0 &gt; C for 2 hours and then to 70 &lt; 0 &gt; C for 16 hours. The solvent was evaporated and the residue was partitioned between water (300 mL) and MTBE (300 mL). The organic layer was separated and washed with water (100 mL, 2 x) and brine (100 mL). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness to give the title compound (75.5 g, 79%). ES / MS (m / e): 309 (M + 1).

Production Example 6

2- (diallylamino) -1- (2-fluorophenyl) ethanone oxime

Diallylamine (1.56 L, 12.2 mol) was added to a solution of 2-bromo-l- (2-fluorophenyl) ethanone (1291 g, 5.8 mol) in ethanol (12.9 L) Lt; / RTI &gt; The reaction mixture was stirred at 22 &lt; 0 &gt; C for 4 hours. Hydroxylamine hydrochloride (543 g, 7.58 mol) was added in portions to the solution and then heated at 70 &lt; 0 &gt; C for 16 hours. The reaction was cooled to room temperature, the solvent was evaporated and the residue was partitioned between water (5.1 L) and MTBE (6.4 L). The aqueous layer was adjusted to pH = 5.5 by addition of sodium carbonate and the aqueous layer was extracted with additional MTBE (1.2 L). The organic layers were combined, washed with water and brine. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness to give the crude title compound (1.45 kg, 104%) which was used without further purification. ES / MS (m / e): 249 (M + 1).

Production Example 7

Benzyl N-allyl-N- [2-hydroxyiminoethyl] carbamate

A solution of benzyl N-allyl-N- (2-oxoethyl) carbamate (25 g, 107 mmol) in acetonitrile (150 mL) was added to a solution of hydroxylamine hydrochloride (9.68 g, 139 mmol ) And sodium acetate trihydrate (16 g, 117.9 mmol). The mixture was stirred at room temperature overnight. The acetonitrile was evaporated and the aqueous solution was extracted with EtOAc. The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo to give the title compound (24 g, 90%). ES / MS (m / e): 249 (M + H).

Production Example 8

2-Bromo-1- (5-bromo-2-fluorophenyl) ethan-

(984 g, 5.53 mol) was added to a solution of l- (5-bromo-2-fluorophenyl) ethan-l-one (1000 g, 4.6 mol) in DCM (7 L) And p-toluenesulfonic acid (1315 g, 7.64 mol) in several portions. The mixture was stirred and heated to 40 &lt; 0 &gt; C. The mixture was cooled to 24 ℃, it was added to _{7% NaHCO 3 (5 L)} . Separate the layers and wash the organic layer with _{10% Na 2 SO 3 (5} L) and water (5 L). The organic layer was concentrated to 2-3 volumes to give the title compound which was used without further purification.

Production Example 9

LH-pyrrolo [3,4-c] isoxazole (2-fluoro-4-methoxybenzyl)

To a solution of 2-bromo-l- (5-bromo-2-fluorophenyl) ethan-l-one (1363 g, 4.61 mol) in toluene (10 L) was added diallylamine (537 g, 5.53 mol) And dipea (2381 g, 18.42 mol). The mixture was stirred at 40 &lt; 0 &gt; C for 4 hours to give l- (5-bromo-2-fluorophenyl) -2- (diallylamino) ethan-l-one which was not isolated. To a mixture of N- (4-methoxybenzyl) hydroxylamine (847 g, 5.53 mol) and Ti (OiPr) ₄ (1965 g, 6.91 mol) - (diallylamino) ethan-1-one. The mixture was stirred at 90 &lt; 0 &gt; C for 2 hours. The mixture was cooled to 20 ℃, was added 50% citric acid monohydrate (4 L) and saturated _{Na 2 CO 3 (4 L)} . The layers were separated and the aqueous layer was extracted with MTBE (5 L). The organic extracts were washed with water (5 L), filtered through diatomaceous earth, and concentrated to dryness. EtOAc (10 L) and oxalic acid (580 g) were added to the residue, and the solid was filtered and added to 1 N NaOH (13 L). MTBE (5 L) was added and the mixture was filtered through diatomaceous earth. The layers were separated and the organic layer was concentrated to 2 volumes. Heptane (3 L) was added and the solution was cooled to 10 &lt; 0 &gt; C. The resulting solid was filtered to give the title compound (1330 g, 64%).

¹ H NMR (400 MHz, CDCl ₃ )?: 2.51-2.49 (m, 3H), 3.09-3.04 (m, 3H), 3.78-3.41 (m, 6H), 4.01 m, 2H), 5.89-5.85 (m, 1H), 6.82-6.80 (m, 2H), 7.51-7.13 (m, 3H), 7.63-7.62 (m,

Production Example 10

(3-bromophenyl) -3,3a, 4,6-tetrahydro-lH-pyrrolo [3,4-c]

The crude 1- (3-bromophenyl) -2- (diallylamino) ethanone oxime (75.5 g, 195.34 mmol) was dissolved in toluene (600 mL) and refluxed for 12 hours. The solvent was evaporated in vacuo and the residue was dissolved in a mixture of aqueous 1 N HCl (1 L) and MTBE (300 mL). The mixture was stirred for 15 minutes and diatomaceous earth (10 g) was added. The mixture was stirred for an additional 20 minutes and filtered through diatomaceous earth. The filter cake was washed with additional aqueous 1 N HCl (200 mL) and MTBE (200 mL). The organic layer was separated and washed with 1 N HCl (2 x 100 mL). The aqueous layers were combined and the pH was adjusted to 9 using 50% w / w NaOH. The aqueous mixture was extracted with MTBE (3 x 250 mL). The organic layers were combined, dried over sodium sulfate and filtered. The filtrate was evaporated and dried under vacuum to give a red solid (60 g). The red solid was diluted with heptane (600 mL) and the mixture was heated under reflux for 20 minutes. Charcoal (2 g) was added and the mixture was filtered through diatomaceous earth. The filtrate was concentrated under atmospheric pressure to adjust the final volume to 300 mL. The solution was cooled to 22 &lt; 0 &gt; C and stirred for 3 hours. The light yellow solid was collected via filtration and dried to constant weight under vacuum to give the title compound (40 g, 60%). ES / MS (m / e): 309 (M + 1).

Production Example 11

3,4-c] isoxazole (2-fluoro-phenyl) -3,3a, 4,6-tetrahydro-lH-pyrrolo [3,4-

Flow Chemical Reaction Step: A 343-ml stainless steel tubular reactor (OD = 1/8 inch) was placed in a GC oven and flushed with toluene at 20 mL / min for 20 minutes. A back pressure of nitrogen (720 psig) was applied and the temperature of the GC was set at 210 占 폚. After the temperature reached 210 캜, a solution of 2- (diallylamino) -1- (2-fluorophenyl) ethanone oxime (480.51 g, 1.74 mol) in toluene (5.81 L) A pair of high pressure syringe pumps were pumped through the reactor at 22.866 mL / min to provide a residence time of 15 minutes. After all the stock solution had been consumed, the reactor was flushed with toluene for 30 minutes at 22.866 mL / min. The temperature of the GC oven was set at 25 DEG C and the total solution was collected and concentrated under vacuum. The solvent was evaporated and the residue was dissolved in methylene chloride (2.5 L) and water (5 L). The pH was adjusted to 1 using hydrochloric acid, the aqueous layer was separated, and the pH was adjusted to 10 by neutralizing with sodium hydroxide. The aqueous layer was extracted with MTBE (3 x 2.5 L). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated to dryness to give the crude title compound (248 g, 47%) which was used without further purification. ES / MS (m / e): 249 (M + 1).

Production Example 12

(5-bromo-2-fluorophenyl) hexahydro-lH-pyrrolo [3,4-c] isoxazole hydrochloride

Trifluoroacetic acid (4 L, 52.9 mol) was added to a solution of 5-allyl-6a- (5-bromo-2-fluorophenyl) -l- (4- methoxybenzyl) hexahydro- lH -Pyrrolo [3,4-c] isoxazole (1990 g, 4.45 mol) at such a rate as to keep the temperature below 35 &lt; 0 &gt; C. After the addition was complete, the mixture was warmed to 33-43 &lt; 0 &gt; C and stirred for 6 hours. NaOH (20%, 10 L) was added at a rate to keep the temperature below 35 &lt; 0 &gt; C. The layers were separated and the organic layer was washed with water (6 L). The solution was concentrated, ethanol (16 L) was added, and the mixture was filtered through diatomaceous earth. The filtrate was concentrated and EtOAc (10 L) was added. 4 M HCl in EtOAc (8 L) was added and the resulting solid was filtered and dried to give the title compound (1385 g, 85.6%). ES m / z 327.1 (M + 1)

Production Example 13

Benzyl 3,3a, 4,6-tetrahydropyrrolo [3,4-c] isoxazole-5-carboxylate

A solution of benzyl N-allyl-N- [2-hydroxyiminoethyl] carbamate (24 g, 96.6 mmol) in DCM (338 mL) was treated with 5% w / w sodium hypochlorite (106.08 mmol, 143.06 mL) Aqueous solution over 10 min. The resulting mixture was stirred at room temperature overnight. The reaction was quenched with 40% aqueous solution of sodium bisulfate (7 g). The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified on silica gel eluting with 5% EtOAc in hexanes to give the title compound (18 g, 75%). ES / MS (m / e): 247 (M + H).

Production Example 14

LH-pyrrolo [3,4-c] isoxazole-5-carboxylate &lt; / RTI &gt;

A solution of n-butyllithium in 1.6 M hexane (25.4 mL, 40.6 mmol) was added to a -78 [deg.] C solution of 4-bromo-1-fluoro-2-iodobenzene (12.22 g, 40.6 mmol) in THF To give a yellow solution, which was stirred for 15 minutes at -78 &lt; 0 &gt; C. (5.14 mL, 40.6 mmol) in THF (60 mL) was added to a solution of benzyl 3,3a, 4,6-tetrahydropyrrolo [3,4-c] isoxazole-5-carboxylate g, 20.3 mmol) and the mixture was stirred at -78 &lt; 0 &gt; C for 5 min. This solution was added via a cannula to a previously prepared &lt; RTI ID = 0.0 &gt; -78 C &lt; / RTI &gt; The combined mixture was stirred at -78 &lt; 0 &gt; C for 30 minutes. The mixture was quenched with saturated aqueous ammonium chloride and warmed to room temperature. The mixture was extracted with EtOAc (3 x), the organic extracts were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified on silica gel with a 5% to 100% EtOAc in 35 min gradient in hexanes to give the title compound (2.27 g, 27%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 421/423 (M + H).

Production Example 15

Benzyl 1- (benzoylcarbamoyl) -6a- (5-bromo-2-fluoro-phenyl) -3,3a, 4,6- tetrahydropyrrolo [3,4- c] isoxazole- - carboxylate

Benzoyl isothiocyanate (2.87 mL, 21.28 mmol) was added to a solution of benzyl 6a- (5-bromo-2-fluoro-phenyl) -3,3a, 4,6-tetrahydro- 3-isoxazole-5-carboxylate (5.977 g, 14.2 mmol) and stirred under nitrogen overnight. The solvent was removed in vacuo. The crude product was purified on a silica gel with a gradient from 5% to 100% EtOAc in hexanes over 30 min to give the title compound (6.05 g, 73%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 584/586 (M + H).

Production Example 16

Benzyl 3- (benzoylcarbamoylamino) -3- (5-bromo-2-fluoro-phenyl) -4- (hydroxymethyl) pyrrolidine-

Benzyl 1- (benzoylcarbamoyl) -6a- (5-bromo-2-fluoro-phenyl) -3,3a, 4,6- tetrahydropyrrolo [3,4- c] isoxazole- -Carboxylate (6.05 g, 10.4 mmol) and zinc (powder, <10 micrometer) (6.77 g, 103.5 mmol) in acetic acid (52 mL) at room temperature overnight under nitrogen. The reaction was diluted with EtOAc and filtered through diatomaceous earth. The solvent was removed in vacuo and the residue was diluted with EtOAc, water, and saturated aqueous sodium bicarbonate. The mixture was extracted with EtOAc (3 x), the combined organic layers were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified on silica gel by gradient from 5% to 100% EtOAc in hexanes over 30 min to give the title compound (5.222 g, 86%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 586/588 (M + H).

Production Example 17

(1-allyl-4-amino-4- (5-bromo-2-fluorophenyl) pyrrolidin-

A saturated aqueous solution of sodium carbonate was added to a solution of 5-allyl-6a- (5-bromo-2-fluorophenyl) hexahydro-lH-pyrrolo [3,4- c] isoxazole hydrochloride (1400 g, , 3.85 mol) to a pH > 9. The layers were separated and the organic extract was concentrated to 1.5 volumes. Acetic acid (1.38 L) was added and the solution was concentrated to 2 L. Acetic acid (7 L) and zinc powder (2.5 kg, 38.5 mol) were added and the mixture was heated to 40-50 &lt; 0 &gt; C and stirred for 3 hours. EtOAc (9.8 L) was added and the mixture was filtered through diatomaceous earth. The filter cake was washed with EtOAc (4 L). The filtrate was separated and water (7 L) was added to the combined organic fractions. Ammonium hydroxide was added to reach pH? 9. The layers were separated and the organic layer was concentrated to 2 L. Ethanol (2.8 L) was added and the solution was concentrated to 2 L. Ethanol (19 L) was added and the mixture was filtered through diatomaceous earth to give an ethanol solution of the title compound which was used without further purification.

Production Example 18

[1-allyl-4-amino-4- (3-bromophenyl) pyrrolidin-3- yl] methanol

To a solution of 5-allyl-6a- (3-bromophenyl) -3,3a, 4,6-tetrahydro-lH-pyrrolo [3,4- c] isoxazole (40 g, 129.4 mmol ) Was treated in one portion with zinc dust (42.3 g, 646.8 mmol). The reaction was stirred vigorously at room temperature for 1 hour. EtOAc (400 mL) was added and the mixture was filtered through diatomaceous earth. The filtrate was evaporated and the residue was dried under vacuum. The residue was partitioned between water (300 mL) and MTBE (300 mL). The pH was adjusted to 8 using 50% w / w sodium hydroxide and the organic layer was separated, dried over sodium sulfate and filtered. The filtrate was evaporated and the residue dried under vacuum to give the title compound (41 g, 97%). ES / MS (m / e): 311 (M + 1).

Production Example 19

4-amino-4- (2-fluorophenyl) pyrrolidin-3-yl] methanol

Zinc dust (590 g, 9 mol) was added to a solution of 5-allyl-6a- (2-fluorophenyl) -3,3a, 4,6-tetra Pyrrolo [3,4-c] isoxazole (3559 g, 1.29 mol) in dichloromethane and the mixture was heated at 70 &lt; 0 &gt; C for 16 hours. The reaction was cooled to 60 C, diluted with THF (2.85 L), and filtered hot on diatomaceous earth. The filtrate was evaporated to remove the organic solvent and the aqueous mixture was diluted with a 10% w / w citric acid aqueous solution (4 L) and EtOAc (3.5 L). The organic layer was separated and the aqueous layer was washed with EtOAc (2 x 2 L). The aqueous layer was neutralized with sodium hydroxide 50% w / w to adjust the pH to 10 and extracted with EtOAc (2 x 1.5 L). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated to dryness to give the crude title compound (299 g, 92%). ES / MS (m / e): 251 (M + 1).

Production example 20

(2S, 3S) - (3S, 4R) -l-allyl-3- (5-bromo-2-fluoro-phenyl) -4- (hydroxymethyl) pyrrolidin- Hydroxy-2,3-bis [(4-methylbenzoyl) oxy] -4-oxo-butanoate

Di-p-toluoyl-L-tartaric acid monohydrate (1.04 kg, 2.69 mol) was added to a solution of (1-allyl- 3-yl) methanol (1264 g, 3.85 mmol) in dichloromethane (5 mL). The mixture was heated to 65-75 &lt; 0 &gt; C and stirred for 3 hours. The mixture was cooled to 5-10 &lt; 0 &gt; C and the seed crystals were dissolved in [(3S, 4R) -l-allyl-3- (5-bromo-2- fluoro-phenyl) -4- (hydroxymethyl) pyrrolidine -3-yl] ammonium; 1.0 g of (2S, 3S) -4-hydroxy-2,3-bis [(4-methylbenzoyl) oxy] -4-oxo-butanoate, And stirred for 3 hours. The solids were filtered and the filter cake was washed with cold ethanol (1.4 L). The filter cake was dried to give the title compound as a white solid. Chiral analysis of the second eluent: Column: IC chiral pack, 4.6 mm * 250 mm * 5 [mu] m; Eluent: 90% hexane (0.3% diethylamine); 10% ethanol (0.3% diethylamine); Enantiomerically enriched (99% ee) enantiomers (1050 g, 38%) were identified at a flow rate of 1.0 mL / min and UV 270 nm for R _t = 7.4 min.

¹ H NMR (400 MHz, CD ₃ OD)?: 2.40 (s, 6H), 3.05-3.04 (m, 1H), 3.57-3.31 (m, 3H), 3.66-3.58 (m, 2H), 5.38-5.36 (m, IH), 5.50-5.46 (m, IH), 5.88 (d, J = 8.0 Hz, 4H), 7.53-7.51 (m, IH), 7.80-7.78 (m, IH), 8.01 (d, J = 8.0 Hz, 4H).

Production Example 21

[(3R, 4S) -1-allyl-4-amino-4- (2-fluorophenyl) pyrrolidin-3-yl] methanol; 2,3-bis [(4-methylbenzoyl) oxy] butanedioic acid

A solution of di-p-toluoyl-L-tartaric acid (348.6 g, 884 mmol) in 1-methoxy-2-propanol (1.13 L) was added dropwise to 40 [deg.] C 1-methoxy- Pyridin-3-yl] methanol (225.9 g, 902 mmol) in anhydrous THF (5 mL). The reaction was cooled to 22 &lt; 0 &gt; C and stirred for 18 hours. The white solid was collected by filtration and washed with 1-methoxy-2-propanol (600 ml). The collected solid was dried to give the title compound (183.01 g, 31.8%). ES / MS (m / e): 251 (M + 1).

Production Example 22

[(3R, 4S) -1-allyl-4-amino-4- (3-bromophenyl) pyrrolidin-3-yl] methanol; (2R, 3R) -2,3-bis [(4-methylbenzoyl) oxy] butanedioic acid

A solution of [1-allyl-4-amino-4- (3-bromophenyl) pyrrolidin-3-yl] methanol (77 g, 235 mmol) in isopropyl alcohol (914 mL) . D-p-Toluyl-L-tartaric acid (86.2 g, 223 mmol) was added and the mixture was cooled to 22 [deg.] C for 2 h and stirred overnight. The slurry was filtered to collect a pale yellow solid and washed with isopropyl alcohol. The solid was dried in vacuo to give the title compound (63 g, 36%). ES / MS (m / e): 311 (M + 1). The product was analyzed by reverse phase chiral chromatography: analysis of the first eluent (column: chiral pack ID-3 4.6 x 50 mm; eluent: 70:30, aqueous 20 mM ammonium bicarbonate: acetonitrile; min, UV 215 nm) has R _t = 1.26 minutes, enantiomeric rich (96% ee by a) was confirmed enantiomer.

Production Example 23

(3R, 4S) -1-allyl-4-amino-4- (5-bromo-2- fluorophenyl) pyrrolidin-

1 N HCl (500 mL, 500 mmol) was added to a solution of [(3S, 4R) -1-allyl-3- (5-bromo- ) Pyrrolidin-3-yl] ammonium; (2S, 3S) -4-hydroxy-2,3-bis [ ) &Lt; / RTI &gt; The mixture was stirred for 1 hour. The aqueous layer was separated and the pH was adjusted to 8 using 1 N NaOH. The aqueous layer was extracted with EtOAc (350 mL x 2). The organic layers were combined, washed with water (500 mL) and concentrated to give the title compound (40 g, 87%). Chiral analysis of the second eluent: Column: IC chiral pack, 4.6 mm * 250 mm * 5 [mu] m; Eluent: 90% hexane (0.3% diethylamine); 10% ethanol (0.3% diethylamine); An enantiomerically enriched (99.7% ee) enantiomer was identified at a flow rate of 1.0 mL / min and UV 270 nm for R _t = 7.4 min.

^{1 H NMR (400 MHz, CDCl} 3 δ: 2.78-2.70 (m, 5H), 3.16-3.00 (m, 3H), 3.87-3.75 (m, 1H), 3.90-3.84 (m, 1H), 5.24-5.11 (m, 2H), 5.91-5.87 (m, IH), 6.95-6.91 (m, IH), 7.35-7.32 (m, IH), 7.67-7.

Production Example 24

4-amino-4- (2-fluorophenyl) pyrrolidin-3-yl] methanol

[(3R, 4S) -1-allyl-4-amino-4- (2- fluorophenyl) pyrrolidin- Acid (211 g, 331 mmol) was dissolved in water (2.1 L) and EtOAc (2.3 L). The pH was adjusted to 1 by adding 35% w / w hydrochloric acid. The aqueous layer was separated and the pH was adjusted to 10 using 50% w / w sodium hydroxide and extracted with EtOAc (2x). The pH of the aqueous layer was adjusted to 10 with aqueous NaOH and the pH of the aqueous solution was maintained at pH = 10 with extraction with MTBE (3x). The organic extracts were combined, dried over sodium sulfate, filtered and concentrated to dryness to give the crude title compound (73 g, 88%, 94.8% ee). The product was purified by chiral chromatography: column AS-H, eluent 10% isopropyl alcohol, 2% isopropylamine; Flow rate 3 mL / min, UV 220; Analysis by pressure of 100 bar at 35 &lt; 0 &gt; C gave the title compound as a second eluent, R _f = 2.26 min. ES / MS (m / e): 251 (M + 1).

Production example 25

((3S, 4R) -1-allyl-3- (5-bromo-2-fluorophenyl) -4- (hydroxymethyl) pyrrolidin- amides

Benzoyl isothiocyanate (15.0 g, 91.9 mmol) was added to a solution of ((3R, 4S) -l-allyl-4-amino- 4- (5-bromo-2- fluorophenyl) 3-yl) methanol (30 g, 91.1 mmol). The solution was warmed to 25 &lt; 0 &gt; C and stirred for 1 hour to give a THF solution of the title compound which was used without further purification.

Production Example 26

4-amino-4- (3-bromophenyl) pyrrolidin-3-yl] methanol

[(3R, 4S) -1-allyl-4-amino-4- (3-bromophenyl) pyrrolidin-3-yl] methanol; (63 g, 85.8 mmol) was combined with aqueous 1 N HCl (800 mL) and EtOAc (400 mL) and the mixture was stirred at 22 &lt; RTI ID = 0.0 &gt; RTI ID = 0.0 &gt; C &lt; / RTI &gt; The layers were separated and the pH of the aqueous layer was adjusted to 10 using 50% w / w sodium hydroxide. The aqueous mixture was extracted with MTBE (3 x 250 mL). The combined organic layers were dried over magnesium sulfate, filtered and evaporated to dryness to give the title compound (27 g, 99%). ES / MS (m / e): 311 (M + 1).

Production Example 27

3,4-d] [l, 3] thiazin-3-yl) - N - [(4aR, 7aS) 2-yl] benzamide

A solution of [(3R, 4S) -l-allyl-4-amino-4- (3-bromophenyl) pyrrolidin-3- yl] methanol (27 g; 86.7 mmol) in THF (270 mL) Lt; RTI ID = 0.0 &gt; -5 C. &lt; / RTI &gt; Benzoyl isothiocyanate (12.3 mL, 91 mmol) was added dropwise while maintaining the temperature below 0 &lt; 0 &gt; C. The reaction was allowed to warm to 22 &lt; 0 &gt; C for 1 hour. 1,1'-Carbonyldiimidazole (28.1 g, 173.5 mmol) was added in one portion and the reaction was stirred at 22 &lt; 0 &gt; C for 1 h and then heated under reflux for 16 h. The solvent was removed in vacuo and the residue was dried under vacuum. The crude material was partitioned between MTBE (500 mL) and water (250 mL). The organic layer was separated, dried over magnesium sulfate, filtered and evaporated to dryness. Purification of the crude material on silica gel with a gradient from 90/10 to 60/40 DCM / EtOAc afforded the title compound (27 g, 68%). ES / MS (m / e): 456 (M + 1).

Production Example 28

N - ((4aR, 7aS) -6-allyl-7a- (5-bromo-2- fluorophenyl) -4,4a, 5,6,7,7a- hexahydropyrrolo [3,4- d ] [1,3] thiazin-2-yl) benzamide, dihydrochloride

(36.8 g, 140.3 mmol) was reacted with N - ((3S, 4R) -1-allyl-3- (5-bromo-2- fluorophenyl) -4- (hydroxymethyl) Pyridin-3-yl) carbamothioyl) benzamide (91.1 mmol) in THF (400 mL). Di-t-butyl azodicarboxylate (31.6 g, 137.2 mmol) in THF (100 mL) was added. The mixture was stirred at 20-30 &lt; 0 &gt; C for 2 hours. The mixture was concentrated and MTBE (400 mL) was added. The solution was filtered through diatomaceous earth and the cake was washed with MTBE (130 mL). The filtrates were combined and 1 N HCl in EtOAc (200 mL) was added. The mixture was stirred for 2 hours and then concentrated to 500 mL. MTBE (320 mL) was added, the solution was filtered and washed with heptane (130 mL). The solid was slurried with EtOAc (650 mL) and stirred at 50-60 [deg.] C for 2 hours. The hot slurry was filtered and the solid was washed with EtOAc (130 mL) and heptane (130 mL). The solids were reslurried in EtOAc (650 mL) and stirred at 50-60 [deg.] C for 2 hours. The hot slurry was filtered and washed with EtOAc (130 mL) and heptane (130 mL). The solid was dried to give the title compound as di-HCl salt (40 g, 80%, 99.5% ee). Chiral analysis of the first eluent: Column: IC Chiralpak, 4.6 mm * 250 mm * 5 [mu] m; Eluent: 85% hexane (0.1% diethylamine): 15% isopropyl alcohol (0.1% diethylamine); Enantiomerically enriched (99.5% ee) enantiomers were identified at a flow rate of 1.0 mL / min and UV 282 nm for R _t = 12.5 min.

Production Example 29

(4aR, 7aS) -6-allyl-7a- (2-fluoro-5- (2,2,2- trifluoroacetamido) phenyl) -4,4a, 7a-hexahydropyrrolo [3,4-d] [1,3] thiazin-2-yl) benzamide

15% sodium carbonate (440 mL) was added to a solution of N - ((4aR, 7aS) -6-allyl-7a- (5-bromo-2- fluorophenyl) 4a, 5,6,7,7a-hexahydropyrrolo [3,4- d] [1,3] thiazin-2-yl) benzamide dihydrochloride (495 g, 717.88 mmol) . The mixture was stirred for 1-2 hours. The layers were separated and the organic layer was filtered through silica gel (40 g) and washed with EtOAc (600 mL). The filtrate was concentrated to dryness to give N - ((4aR, 7aS) -6-allyl-7a- (5-bromo-2- fluorophenyl) -4,4a, 5,6,7,7a-hexahydropyrrolo [3,4-d] [1,3] thiazin-2-yl) benzamide was obtained. Acetamide (136.7 g, 1.21 mol), NaI (182.5 g, 1.22 mol), 4 A molecular sieves (342 g), and _{K 2 CO 3 (170.9 g,} 1.24 mol) in trifluoroacetic DMSO (525 mL) and To a solution of N - ((4aR, 7aS) -6-allyl-7a- (5-bromo-2- fluorophenyl) -4,4a, 5,6,7,7a -Hexahydro- pyrrolo [3,4-d] [1,3] thiazin-2-yl) benzamide (341 g, 494.54 mmol). Trans-N, N'-dimethylcyclohexane (81.6 g, 573.66 mmol) and copper iodide (27.3 g, 143.34 mmol) in DMSO (500 mL) were added to the reaction mixture. The mixture was stirred for 5 minutes. The mixture was warmed to 100 占 폚, stirred for 8 hours, and cooled to 24 占 폚. Water (5.9 L) and DCM (5.9 L) were added, the mixture was filtered, and the layers were separated. The organic layer was washed with water (5.9 L) to give the title compound in a solution of DCM which was used without further purification.

Production Example 30

3,4a, 5,6,7,7a-hexahydropyrrolo [3,4-d] pyrimidin-2-one, N - ((4aR, 7aS) [1,3] thiazin-2-yl) benzamide, hydrochloride

Sodium hydroxide (28.7 g) and water (2.7 L) were added to a solution of N - ((4aR, 7aS) -6-allyl-7a- (2-fluoro-5- (2,2,2- trifluoroacetamido) Phenyl) -4,4a, 5,6,7,7a-hexahydropyrrolo [3,4- d] [1,3] thiazin-2-yl) benzamide (250 g, 494.4 mmol) And the mixture was stirred at 24 &lt; 0 &gt; C for 68 hours. 1 N HCl (3.5 L) was added to obtain a pH of 1-3. The layers were separated and the aqueous layer was washed with DCM (680 mL). DCM (4 L) was added to the aqueous portion followed by 21% ammonium hydroxide to obtain a pH of 8-10. The layers were separated, the organic extracts were combined, filtered through silica gel (170 g) and washed with DCM (1.4 L). The solvent was concentrated to dryness and diluted with EtOAc (4 L). 1 N HCl in EtOAc (700 mL) was added at a temperature below 25 &lt; 0 &gt; C, and the mixture was stirred for 1 hour. The mixture was concentrated to about 7-8 volumes and EtOAc (2.8 L) was added. The resulting precipitate was filtered and washed with EtOAc (400 mL). The solid was dried to give the title compound (246 g, 52%).

Production Example 31

N - ((4aR, 7aS) -7a- (5-Acetamido-2-fluorophenyl) -6-allyl-4,4a, 5,6,7,7a- hexahydropyrrolo [ d] [l, 3] thiazin-2-yl) benzamide

Acetic anhydride (23.5 g, 0.23 mol) was added to a solution of N - ((4aR, 7aS) -6-allyl-7a- (5-amino-2- fluorophenyl) -4,4a, Benzamide hydrochloride (100 g, 0.153 mol) and triethylamine (54.3 g, 0.535 mol) in tetrahydrofuran (5 mL) Lt; / RTI &gt; After stirring at 20-25 ° C for 1 h, saturated NaHCO ₃ (700 mL) and water (600 mL) were added. The layers were separated to give the title compound, which was used without further purification as a solution in DCM.

Production example 32

3,4-d] [l, 3] thiazin-3-yl) - N - [(4aR, 7aS) 2-yl] benzamide

To a solution of [(3R, 4S) -1-allyl-4-amino-4- (2- fluorophenyl) pyrrolidin-3- yl] methanol (129.7 g, 414 mmol) in THF The solution was cooled at 0 &lt; 0 &gt; C under a nitrogen atmosphere. Benzoyl isothiocyanate (61.5 mL, 456 mmol) was added maintaining the temperature below 5 &lt; 0 &gt; C. The reaction was warmed to room temperature over 3 hours, 1,1'-carbonyldiimidazole (87.4 g, 538.9 mmol) was added and the reaction stirred at 22 ° C for 1 hour and then at 70 ° C for 16 hours And heated. The reaction mixture was cooled to 22 &lt; 0 &gt; C and the solvent was evaporated. The residue was partitioned between EtOAc (1 L) and water (1 L). The organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 400 mL). The organics were combined, dried over sodium sulfate, filtered and evaporated to dryness to give the crude title compound. The crude product was purified by silica gel chromatography eluting with a gradient of 0-40% DCM to EtOAc / DCM to give the title compound as a light yellow solid (170 g, 99%) containing residual solvent. ES / MS (m / e): 396 (M + 1).

Production Example 33

Benzyl 2-benzamido-7a- (5-bromo-2-fluoro-phenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] 6-carboxylate

1,1'-carbonyldiimidazole (2.87 g, 17.7 mmol) was added to a solution of benzyl 3- (benzoylcarbamoylaminoamino) -3- (5-bromo-2-fluoro- ) -4- (hydroxymethyl) pyrrolidine-1-carboxylate (5.198 g, 8.86 mmol). The mixture was stirred at room temperature for 1.5 hours and then the reaction was heated at reflux under nitrogen overnight. The reaction was cooled, diluted with water and extracted with EtOAc (3 x). The organic layers were combined, dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified on a silica gel with a gradient from 5% to 100% EtOAc in hexanes over 30 min to give the title compound (2.93 g, 58%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br). 568/570 (M + H) &lt;

Production example 34

N - ((4aR, 7aS) -7a- (5-acetamido-2-fluorophenyl) -4,4a, 5,6,7,7a- hexahydropyrrolo [3,4- d] [1 , 3] thiazin-2-yl) benzamide

(4aR, 7aS) -7a- (5-acetamido-2-fluoro-pyrimidin-4-ylamino) triphenylphosphine (4.0 g, 0.015 mol) and 1,3-dimethylbarbituric acid Phenyl) -6-allyl-4,4a, 5,6,7,7a-hexahydropyrrolo [3,4-d] [1,3] thiazin- 2- yl) benzamide (0.153 mol) Solution. Palladium acetate (1.7 g, 7.7 mmol) was added and the mixture was stirred at 20-30 &lt; 0 &gt; C for 1 h. 25% ammonium hydroxide was added and the layers were separated. The organic layer was washed with HOAc (3.0 eq. In 500 mL water) and the pH was adjusted to 8-9 using 25% ammonium hydroxide. The aqueous layer was extracted with DCM (2 x 500 mL). The organic extracts were combined and concentrated to 3-4 volumes. MTBE (1 L) was added and the mixture was filtered. The mixture was concentrated and heptane (1 L) was added. The resulting solid was filtered, collected and dried to give the title compound (48 g, 76%).

^{1 H NMR (400 MHz, CDCl} 3) δ: 2.15 (s, 3H), 2.87-2.83 (m, 1H), 3.43-3.23 (m, 5H), 3.70-3.67 (m, 1H), 7.12-7.07 ( m, 1H), 7.28-7.27 (m, 1H), 7.52-7.41 (m, 4H), 7.79 (m, 1H), 8.18-8.16 (m, 2H). ES m / z 413.1 (M + 1).

Production example 35

Pyrrolo [3,4-d] [l, 3] thiazin-2-ylmethyl) - N - [(4aR, 7aS) - yl] benzamide

To a solution of N - [(4aR, 7aS) -6-allyl-7a- (3-bromophenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] [1 (1 g, 2.19 mmol) and N, N-dimethylbarbituric acid (0.868 g, 5.48 mmol) was bubbled through the resulting slurry with nitrogen at room temperature Degassed for 5 minutes. The mixture was treated with tetrakis (triphenylphosphine) palladium (0.261 g, 219 [mu] mol) and stirred under nitrogen for 1.5 h. In a separate flask, a solution of N - [(4aR, 7aS) -6-allyl-7a- (3-bromophenyl) -4,4a, 5,7-tetrahydropyrrolo [3,4- d] [1,3] thiazin-2-yl] benzamide (22.2 g, 48.6 mmol) and N, N-dimethylbarbituric acid (19.28 g, 121.6 mmol) Nitrogen was bubbled through and degassed. The mixture was treated with tetrakis (triphenylphosphine) palladium (5.79 g, 4.86 mmol) and stirred under nitrogen for 2 hours. The two reactants were combined and the solvent was removed in vacuo to give the crude product. The crude material was purified on silica gel with a gradient of 0.5% to 10% methanol in DCM over 30 min to give the title compound (22.4 g, 100%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 416/418 (M + H).

Production Example 36

Pyrrolo [3,4-d] [l, 3] thiazin-2-one [ - yl] benzamide

(122 g, 793 mmol), bis (dibenzylideneacetone) palladium (4.15 g, 7.21 mmol) and 1,4-bis (diphenylphosphino) butane (3.14 g, 7.21 mmol ) Was added to a solution of N - [(4aR, 7aS) -6-allyl-7a- (2-fluorophenyl) -4,4a, 5,7- tetrahydro- pyrrolo [ 3,4-d] [l, 3] thiazin-2-yl] benzamide (178.21 g, 360 mmol) under nitrogen atmosphere. The solution was degassed three times by a vacuum / nitrogen cycle and then nitrogen was bubbled through the reactants for 15 minutes. Nitrogen was bubbled through the reactants while the reaction mixture was heated to 40 &lt; 0 &gt; C. When the reaction reached 40 캜, the bubbling was removed and the reaction mixture was stirred at 40 캜 for 3 hours under a nitrogen atmosphere. The reaction was cooled to 22 &lt; 0 &gt; C and diluted with water (2 L). The pH was adjusted to 1 by adding a solution of HCl (5 M). The aqueous layer was separated and washed with additional EtOAc (2 x 800 mL). The pH of the aqueous layer was adjusted to 10 using 50% w / w sodium hydroxide and extracted with EtOAc (10 L). The aqueous layer was washed with additional EtOAc (2 x 750 mL). The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to dryness to give the crude title compound as a light yellow solid (124.7 g, 97%). ES / MS (m / e): 356 (M + 1).

Production example 37

Pyrrolo [3,4-d] [l, 3] thiazin-2-one [ - yl] benzamide

Iodotrimethylsilane (2.21 mL, 15.46 mmol) was added to a solution of benzyl 2-benzamido-7a- (5-bromo-2-fluoro-phenyl) -4,4a, Was added dropwise to a room temperature solution of tetrahydropyrrolo [3,4-d] [1,3] thiazine-6-carboxylate (2.93 g, 5.15 mmol). The reaction was stirred at room temperature for 2 hours and the solvent was removed in vacuo. The crude product was purified by SCX column with 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol to give the title compound (2.098 g, 94%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 434/436 (M + H).

Production Example 38

(4aR, 7aS) -2-benzamido-7a- (3-bromophenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] [l, 6-carboxylate

3,4-d] [1, &lt; RTI ID = 0.0 &gt; Butyl dicarbonate (8.81 g, 40.36 mmol) followed by triethylamine (7.67 mL, 55.04 mmol) was added to a room temperature solution of 3-thiazol-2-yl] And the reaction was stirred at room temperature under nitrogen for 1 hour. The solvent was removed in vacuo and the crude product purified on silica gel with a gradient from 5% to 100% EtOAc in hexanes over 25 min to give the title compound (20.22 g, 100%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 516/518 (M + H).

Production example 39

tert-Butyl 2-benzamido-7a- (5-bromo-2-fluoro-phenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] 6-carboxylate

Di-t-butyl dicarbonate (1.16 g, 5.31 mmol) and triethylamine (1.01 mL, 7.25 mmol) were added to a solution of N- [7a- (5- ) -4a, 5,6,7-tetrahydro-4H-pyrrolo [3,4-d] [1,3] thiazin-2- yl] benzamide (2.098 g, 4.83 mmol) . The reaction was stirred under nitrogen at room temperature for 1 hour. The solvent was removed in vacuo and the crude product purified on silica gel with a gradient from 5% to 100% EtOAc in hexanes over 30 min to give the title compound (2.556 g, 99%). ES / MS (m / e): ( ⁷⁹ Br / ⁸¹ Br) 534/536 (M + H).

Production example 40

3,4a, 5,7-tetrahydropyrrolo [3,4-d] [l, 3] thiazin e) -6-carboxylate

To a solution of tert-butyl (4aR, 7aS) -2-benzamido-7a- (3-bromophenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] A solution of trans-N, N'-dimethyl-1,2-cyclohexanediamine (220.3 mg, 1.5 mmol) in acetonitrile (5 g, 9.7 mmol) Was treated with sodium (1.30 g, 19.4 mmol). An aqueous solution of L-ascorbic acid sodium salt (0.66 M, 3.2 mL, 2.1 mmol) and water (10 mL) was added and the top of the flask was purged with nitrogen. The mixture was treated with an aqueous solution of copper (II) sulfate pentahydrate (0.33 M, 3.2 mL, 1.1 mmol) and the mixture was immediately heated on a preheated hot plate at 80 <0> C for 1.5 h under nitrogen. The homogeneous mixture was obtained upon heating. The reaction was cooled and ice water was added. The mixture was extracted with EtOAc (3x). The organic layers were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo to give the crude azide product. The crude azide product was combined with 10% palladium on carbon (2 g) in cold ethanol (150 mL) and the mixture was purged with vacuum / nitrogen followed by vacuum / hydrogen. The mixture was stirred under 30 psi of hydrogen at room temperature for 2 hours. The reaction was drained and the mixture was filtered through diatomaceous earth using DCM to rinse the filter cake. The solvent was removed from the filtrate in vacuo and the crude product was purified on silica gel with 50% EtOAc in DCM to give the title compound (4 g, 91%). ES / MS (m / e): 453 (M + H).

Production example 41

3,4-dihydropyrrolo [3,4-d] [l, 3] thiazin e) -6-carboxylate

To a solution of tert-butyl 2-benzamido-7a- (5-bromo-2-fluoro-phenyl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] Carboxylate (2.556 g, 4.8 mmol) and trans-N, N'-dimethyl-1,2-cyclohexanediamine (150 mg, 1.1 mmol) in acetonitrile Was treated with sodium (933 mg, 14.3 mmol). An aqueous solution of sodium L-ascorbate (0.66 M, 3.2 mL, 2.1 mmol) and water (1 mL) was added and the top of the flask was purged with nitrogen. The mixture was treated with an aqueous solution of copper (II) sulfate pentahydrate (0.33 M, 3.2 mL, 1.1 mmol) and the mixture was immediately heated on a preheated hot plate at 80 <0> C for 1.5 h under nitrogen. The homogeneous mixture was obtained upon heating. The reaction was cooled, diluted with ice water, and the mixture was extracted with EtOAc (3 x). The organic extracts were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo to give the crude azide product. The crude azide product was combined with 10% palladium on carbon (1 g) in cold ethanol (150 mL) and the mixture was purged with vacuum / nitrogen followed by vacuum / hydrogen. The mixture was stirred under 30 psi of hydrogen at room temperature for 5 hours. The reaction was drained, filtered through diatomaceous earth, and the filter cake was rinsed with DCM. The solvent was removed from the filtrate under vacuum and the crude product was purified on silica gel with 50% EtOAc in DCM to give the title compound (2.014 g, 89%). ES / MS (m / e): 471 (M + H).

Production example 42

(4aR, 7aS) -2-benzamido-7α- [3 - [(5-fluoropyridine-2- carbonyl) amino] phenyl] -4,4a, 5,7- tetrahydropyrrolo [3,4-d] [l, 3] thiazine-6-carboxylate

To a solution of tert-butyl (4aR, 7aS) -7a- (3-aminophenyl) -2-benzamido-4,4a, 5,7-tetrahydrocarbodiimide in DCM (4 mL) containing dimethylformamide 5-fluoropyridine-2-carboxylic acid (31.9 mg, 0.23 mmol), 1 &lt; RTI ID = 0.0 & -Dihydroxybenzotriazole hydrate (56.7 mg, 0.41 mmol) and EDCI (40 mg, 0.21 mmol) was treated with DIPEA (179.2 μL, 1.03 mmol) and the resulting mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM (5 mL) and saturated aqueous sodium bicarbonate (15 mL). The organic layer was separated, washed with saturated aqueous sodium chloride (10 mL), dried over sodium sulfate, filtered and the solvent removed in vacuo to afford the title compound (105 mg, 89%). ES / MS (m / e): 576 (M + H).

Production Example 43

Pyrrolo [3,4-d] [l, 3] thiazin-7-ylamino) -4a, 5,6,7-tetrahydro-4H-pyrrolo [3,4- Yl] phenyl] -5-fluoro-pyridine-2-carboxamide; 2,2,2-Trifluoroacetic acid

(4aR, 7aS) -2-benzamido-7α- [3 - [(5-fluoropyridine-2- carbonyl) amino] phenyl] -4,4a, 5,7- tetrahydropyrrolo [3,4-d] [l, 3] thiazine-6-carboxylate (105 mg, 0.18 mmol) was dissolved in DCM (2 mL) and treated with trifluoroacetic acid (500 μL, 6.6 mmol) . The resulting yellow solution was stirred at room temperature for 4 hours and the solvent was removed in vacuo to give crude title product (190 mg, 100%). ES / MS (m / e): 476 (M + H).

Production example 44

Pyrrolo [3,4-d] [l, 3] thiazin-2-ylmethyl) -4H-pyrrolo [ Yl] benzamide

Trifluoroacetic acid (25 mL) was added to a solution of tert-butyl (4aR, 7aS) -7a- (3-aminophenyl) -2-benzamido-4,4a, 5,7-tetrahydrofuran (4 g, 8.84 mmol) in dichloromethane (5 ml) and the mixture was stirred under nitrogen at room temperature for 4 hours. The solvent was removed in vacuo and the crude product was purified by SCX column using 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol to give the title compound (2.49 g, 80%). ES / MS (m / e): 353 (M + H).

Production example 45

Pyrrolo [3,4-d] [l, 3] thiazin-2-yl) -2H- pyrrolo [2,3- Yl] benzamide

Trifluoroacetic acid (10 mL) was added to a solution of tert-butyl 7a- (5-amino-2-fluoro-phenyl) -2-benzamido-4,4a, 5,7-tetrahydrofuran 4-d] [l, 3] thiazine-6-carboxylate (2.013 g, 4.28 mmol) and the mixture was stirred at room temperature under nitrogen for 4 hours. The solvent was removed in vacuo and the crude product was purified by SCX column with 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol to give the title compound (1.555 g, 98%). ES / MS (m / e): 371 (M + H).

Production example 46

N- (4aR, 7aS) -7a- (3-aminophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ d] [l, 3] thiazin-2-yl] benzamide

To a solution of N - [(4aR, 7aS) -7a- (3-aminophenyl) -4a, 5,6,7-tetrahydro-4H-pyrrolo [3,4- d 2-yl] benzamide (2.49 g, 7.06 mmol), 5-fluoro-2-chloropyrimidine (3.74 g, 28.26 mmol) and DIPEA (6.16 mL, 35.32 mmol) Was heated at reflux under nitrogen for 4 hours. The reaction was cooled, diluted with water and extracted with EtOAc (3 x). The combined organic extracts were dried over sodium sulfate, filtered and the solvent removed in vacuo to give the crude product. The crude product was purified on a silica gel with a gradient from 5% to 100% EtOAc in hexanes over 25 min to give the title compound (2.51 g, 79%). ES / MS (m / e): 449 (M + H).

Production example 47

N - [(4aR, 7aS) -7a- (2- fluorophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [3,4 -d] [1,3] thiazin-2-yl] benzamide

To a solution of N - [(4aR, 7aS) -7a- (2-fluorophenyl) -4a, 5,6,7-tetrahydro-4H- pyrrolo [3,4- d] [1,3] thiazin-2-yl] benzamide (124.7 g, 256 mmol), DIPEA (67 mL) and 5-fluoro-2-chloropyrimidine (29.3 ml, 307 mmol) 0.0 &gt; 100 C &lt; / RTI &gt; for 16 hours. The reaction was cooled to 22 &lt; 0 &gt; C and injected into water (10 L) cooled to 10 &lt; 0 &gt; C while maintaining a temperature below 15 &lt; 0 &gt; C. The light cream solid was collected by filtration and washed with additional water. The wet solids were dissolved in EtOAc (2 L) and transferred to a separator funnel. A 5% w / w aqueous solution of sodium chloride (1 L) was added and the organic layer was separated, dried over sodium sulfate, filtered and the filtrate was evaporated under reduced pressure. The product was purified by silica gel chromatography using a gradient of 0-40% EtOAc / isohexane to give the title compound as a light yellow solid (116 g, 70%). ES / MS (m / e): 452 (M + 1).

Production Example 48

N - ((4aR, 7aS) -7a- (5-acetamido-2-fluorophenyl) -6- (5-fluoropyrimidin- 7a-hexahydropyrrolo [3,4-d] [1,3] thiazin-2-yl) benzamide

2-Chloro-5-fluoropyrimidine (28.9 g, 218 mmol) and potassium carbonate (33.46 g, 242.1 mmol) were added to a solution of N - ((4aR, 7aS) -7a- (5- (2-fluorophenyl) -4,4a, 5,6,7,7a-hexahydropyrrolo [3,4-d] [1,3] thiazin- 121.22 mmol). The mixture was heated to 80-85 [deg.] C for 8 hours. The mixture was cooled to 24 &lt; 0 &gt; C, filtered and washed with DMF (100 mL). The solid was slurried with water (2 L) and filtered to give the title compound (68.5 g, 98%). LC-MS: m / z = 509.2 (M + 1) &lt; + &gt;.

^{1 H NMR (400 MHz, d6} -DMSO) δ ppm 1.22 (t, J = 7.28 Hz, 2 H) 1.92 - 2.07 (m, 6 H) 2.89 - 3.20 (m, 2 H) 3.36 - 3.44 (m, 1 H), 3.67 (t, J = 9.54 Hz, 1H) 3.84 (br s, 1H) 4.16 (br s, 2H) 8 H) 7.77 (br s, 2 H) 7.85 - 8.18 (m, 4 H) 8.48 (s, 4 H) 10.15 (br s, 1 H) 10.46 - 10.59 (m, 1H).

Production Example 49

(4aR, 7aS) -7a- (5-Amino-2-fluorophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,6,7,7a-hexahydropyridine 3, 4-d] [l, 3] thiazin-2-amine

Lithium hydroxide (8.6 g, 204.9 mmol) was added to a solution of N - ((4aR, 7aS) -7a- (5-acetamido- 2- fluorophenyl) -6- (5-fluoropyrimidine 3,4-d] [1,3] thiazin-2-yl) benzamide (80 g, 157.3 mmol) Lt; / RTI &gt; The mixture was heated to 60-70 [deg.] C for 4 hours. Concentrated HCl (132 g) was added and the mixture was stirred at 55 &lt; 0 &gt; C for 18 hours. The mixture was cooled to 30 &lt; 0 &gt; C and concentrated to remove methanol. Water was added and the aqueous layer was extracted with DCM (3 x) to give the title compound as an aqueous solution of 920 g of the title compound, 5.6% of the total mass, which was used without further purification.

Production Example 50

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- d] [1,3] thiazin-7-yl] phenyl] -5-fluoro-pyridine-

Pyrrolo [3,4-d] [l, 3] thiazin-7-ylamino) -4a, 5,6,7-tetrahydro-4H-pyrrolo [3,4- Yl] phenyl] -5-fluoro-pyridine-2-carboxamide; (98 μL, 56 μmol) of 2,2,2-trifluoroacetic acid (150 mg, 254 μmol), 5-fluoro-2-chloropyrimidine (68 mg, 51 μmol) and DIPEA was dissolved in DMSO mL) overnight at 40 &lt; 0 &gt; C. Additional 5-fluoro-2-chloropyrimidine (68 mg, 51 μmol) and DIPEA (98 μL, 56 μmol) were added and the mixture was heated at 50 ° C. overnight. Additional 5-fluoro-2-chloropyrimidine (68 mg, 51 μmol) and DIPEA (98 μL, 56 μmol) were added and the mixture was heated overnight at 50 ° C. for the third night. The reaction was cooled and diluted with saturated aqueous sodium carbonate (50 mL) to give a slurry which was filtered and dried in a vacuum oven at 50 &lt; 0 &gt; C for 4 hours to give the title compound (60 mg, 41%). ES / MS (m / e): 449 (M + H).

Alternative Preparation Example 50

N- (4aR, 7aS) -7a- (3-aminophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ d] [1,3] thiazin-2-yl] benzamide (282 mg, 628.73 μmol) and 5-fluoropyridine-2- carboxylic acid (106.46 mg, 754.47 μmol) Formamide &lt; / RTI &gt; (0.5 mL). HOBT (112.70 mg, 817.35 μmol) followed by EDCI (159.07 mg, 817.35 μmol) was added and the resulting mixture was stirred at room temperature for 5 hours under nitrogen. The reaction mixture was diluted with water and the pH was adjusted to ~ 12 with 1 N NaOH. The mixture was extracted with EtOAc (3 x). The organic extracts were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo to give the crude product. The crude product was purified on silica gel with a 20% gradient in 5% to 100% EtOAc in hexanes to give the title compound (327 mg, 91%). ES / MS (m / e): 571 (M + H).

Production example 51

N- [7a- (5-Amino-2-fluoro-phenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ d] [l, 3] thiazin-2-yl] benzamide

Pyrrolo [3,4-d] [l, 3] thiazin-2-yl) -2H- pyrrolo [2,3- A solution of 1-benzoyl benzamide (705 mg, 1.90 mmol), 5-fluoro-2-chloropyrimidine (1.01 g, 7.61 mmol) and DIPEA (1.66 mL, 9.52 mmol) mL) and refluxed under nitrogen for 4 hours. The reaction was cooled, diluted with water and extracted with EtOAc (3 x). The organic layers were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo to give the crude product. The crude product was purified on a silica gel with a gradient from 5% to 100% EtOAc in hexanes over 25 min to afford the title compound (590 mg, 66%). ES / MS (m / e): 467 (M + H).

Production example 52

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- d] [1,3] thiazin-7-yl] phenyl] -5-methoxy-pyrazine-2-carboxamide

N- (4aR, 7aS) -7a- (3-aminophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ yl] benzamide (400 mg, 891.81 μmol) and 5-methoxypyrazine-2-carboxylic acid (165 mg, 1.07 mmol) were dissolved in DCM (4 mL) Formamide &lt; / RTI &gt; (0.5 mL). HOBt (160 mg, 1.16 mmol) followed by EDCI (226 mg, 1.16 mmol) was added and the resulting mixture was stirred at room temperature under nitrogen for 5 hours. The reaction mixture was diluted with water and the pH was adjusted to ~ 12 with 1 N NaOH. The mixture was extracted with EtOAc (3 x). The combined organic extracts were dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified on silica gel with a 20% gradient in 5% to 100% EtOAc in hexanes to give the title compound (482 mg, 92%). ES / MS (m / e): 585 (M + H).

Production Example 53

Dihydro-pyrrolo [3,4-d] [1,3] thiazepin-2-yl) ] Thiazin-7-yl] -4-fluoro-phenyl] -5-fluoro-pyridine- 2- carboxamide

N- [7a- (5-Amino-2-fluoro-phenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ d] [1,3] thiazin-2-yl] benzamide (302 mg, 647 μmol) and 5-fluoropyridine-2-carboxylic acid (110 mg, 777 μmol) Formamide &lt; / RTI &gt; (0.5 mL). HOBT (116 mg, 842 μmol) was followed by EDCI (164 mg, 842 μmol) and the mixture was stirred at room temperature overnight under nitrogen. The reaction mixture was diluted with water, the pH was adjusted to ~ 12 with 1 N NaOH and then extracted with EtOAc (3 x). The organic layers were combined and filtered to collect insoluble material. The solid was washed with water and EtOAc and dried in vacuo to give the title compound. The organic layer from the filtrate was dried over sodium sulfate, filtered and the solvent was removed in vacuo. The residue was purified on silica gel with a gradient of 5% to 100% EtOAc in hexanes over 20 min to give the combined additional title compound (275 mg, 72%). ES / MS (m / e): 590 (M + H).

Production example 54

(5-amino-2-fluoro-phenyl) -6- (3,4-d] [1,3] thiazin-2-yl] benzamide, (Isomer 1)

(5-Amino-2-fluoro-phenyl) -6- (5-fluoropyrimidin-2-yl) -4,4a, 5,7- tetrahydropyrrolo [ 3-yl] benzamide (1.694 g, 3.63 mmol) was separated by chiral HPLC (column: Chiralcel OJ, 8 x 35 cm; eluent: 90% % Dimethylethylamine) and 10% acetonitrile, flow rate 400 mL / min, UV 280 nm). Analysis of the first eluent (column: chiralcel OJ-H 0.46 x 15 cm; eluent: 10:90 acetonitrile: methanol with 0.2% dimethylethylamine; flow rate: 0.6 mL / min, UV 280 nm) Enantiomerically enriched (99% ee) enantiomers (723 mg, 43%) were identified at _t = 6.70 min. ES / MS (m / e): 467 (M + H).

Production example 55

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide, (isomer 1)

(5-amino-2-fluoro-phenyl) -6- Yl] benzamide, isomer 1 (0.361 g, 0.77 mmol) was dissolved in a mixture of DCM (4 mL) and DMF (0.5 mL). 5-methoxypyrazine-2-carboxylic acid (240 mg, 1.55 mmol), HOBT (210 mg, 1.55 mmol) and EDCI (300 mg, 1.55 mmol) were added to the mixture and the mixture was stirred overnight at room temperature. The reaction solution was added directly onto a 12 g silica gel loading column and purified by using a 40 g silica gel column and eluting with a 0-100% EtOAc / Hexane gradient. The product was dissolved in EtOAc (200 mL), washed with 1 N NaOH (2 x 50 mL), and brine (1 x 50 mL). The silica gel tablets were repeated as described above to give the title compound (350 mg, 74%). ES / MS (m / e): 603 (M + H).

Production example 56

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- d] [1,3] thiazin-7-yl] phenyl] -5-cyano-pyridine-

N- (4aR, 7aS) -7a- (3-aminophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ yl] benzamide (0.30 g, 0.67 mmol) was dissolved in DCM (10 mL) and 5-cyanopyridine-2-carboxylic acid (129 mg, 0.87 mmol) And HOBt (185 mg, 1.34 mmol) and EDCI (169 mg, 0.87 mmol) were added. DIPEA (0.35 mL, 2 mmol) was added and the reaction was stirred overnight at room temperature. The material was purified directly by silica gel chromatography, eluting with a 0-100% EtOAc / hexanes gradient to give the title compound (360 mg, 88%). ES / MS (m / e): 579 (M + H).

Production Example 57

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- d] [1,3] thiazin-7-yl] phenyl] -3,5-difluoro-pyridine-2- carboxamide

N- (4aR, 7aS) -7a- (3-aminophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [ yl] benzamide (0.30 g, 0.67 mmol) was dissolved in DCM (10 mL) and 3,5-difluoropyridine-2-carboxylic acid (138 mg, 0.87 mmol ) And HOBT (185 mg, 1.34 mmol) and EDCI (169 mg, 0.87 mmol) were added. Dipea (0.35 mL, 2 mmol) was added and the reaction was stirred overnight at room temperature. The reaction was purified directly by silica gel chromatography, eluting with a 0-100% EtOAc / hexanes gradient to give the title compound (330 mg, 84%). ES / MS (m / e): 590 (M + H).

Production Example 58

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- yl] -4-fluoro-phenyl] -5-cyano-pyridine-2-carboxamide, (isomer 1)

(5-amino-2-fluoro-phenyl) -6- 2-yl] benzamide (0.180 g, 0.39 mmol, isomer 1) was dissolved in a mixture of DCM (2 mL) and DMF (0.25 mL). 5-cyanopyridine-2-carboxylic acid (114 mg, 0.77 mmol), HOBt (106 mg, 0.77 mmol) and EDCI (150 mg, 0.77 mmol) were added and the reaction was stirred overnight at room temperature. The mixture was diluted with water (10 mL), EtOAc (10 mL) and added to a solution of 1 N NaOH (100 mL). The mixture was extracted with EtOAc (2 x 100 mL), the organic layers were combined and washed with brine. The organic layer was dried over MgSO _4, filtered, and concentrated. The residue was purified on silica gel chromatography using a 0-100% EtOAc / hexanes gradient to give the title compound (133 mg, 57%). ES / MS (m / e): 597 (M + H).

Production example 59

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- yl] -4-fluoro-phenyl] -3,5-difluoro-pyridine-2-carboxamide, (isomer 1)

(5-amino-2-fluoro-phenyl) -6- 2-yl] benzamide (0.180 g, 0.39 mmol, isomer 1) was dissolved in a mixture of DCM (2 mL) and DMF (0.25 mL). 5-cyanopyridine-2-carboxylic acid (114 mg, 0.77 mmol), HOBt (106 mg, 0.77 mmol) and EDCI (150 mg, 0.77 mmol) were added and the reaction was stirred overnight at room temperature. The mixture was diluted with water (10 mL) and EtOAc (10 mL) and then poured into a solution of 1 N NaOH (100 mL). The mixture was extracted with EtOAc (2 x 100 mL), the organic extracts were combined and washed with brine. The organic layer was dried over MgSO _4, filtered, and concentrated. The residue was purified by silica gel chromatography using a 0-100% EtOAc / hexanes gradient to give the title compound (190 mg, 80%). ES / MS (m / e): 608 (M + H).

Production example 60

(4aR, 7aS) -7a- (2-fluorophenyl) -6- (5-fluoropyrimidin- 2- yl) -4,4a, 5,7- tetrahydropyrrolo [3,4- d] [L, 3] thiazin-2-amine

Lithium hydroxide (9.26 g, 386 mmol) was added to a solution of N - [(4aR, 7aS) -7a- (2- fluorophenyl) -6- (5-fluoropyrimidin- Was added to a mixture of 4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-2-yl] benzamide (158.6 g, 351.6 mmol). The mixture was heated at 70 &lt; 0 &gt; C for 4 hours and then cooled to 22 &lt; 0 &gt; C. The reaction mixture was evaporated in vacuo to a yellow residue. The residue was partitioned between water (1 L) and EtOAc (750 mL). HCl (5 M aqueous solution) was added to adjust the pH to 1. The aqueous layer was separated and the organic layer was washed with EtOAc (2 x 200 mL). The pH of the aqueous layer was adjusted to pH = 10 using a 50% w / w aqueous solution of sodium hydroxide and extracted with EtOAc (3 x 1 L). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude title compound as a light yellow solid (133.3 g, 99%, with 12% residual EtOAc). ES / MS (m / e): 348 (M + 1).

Production example 61

(4-amino-2-fluoro-phenyl) -6- (5-fluoropyrimidin- , 4-d] [l, 3] thiazin-2-amine

Sulfuric acid (33.4 ml, 626.6 mmol) was added to a solution of (4aR, 7aS) -7a- (2-fluorophenyl) -6- (5-fluoropyrimidin- , 4a, 5,7-tetrahydropyrrolo [3,4-d] [l, 3] thiazin-2-amine (45.8 g, 125.3 mmol). The mixture was cooled to 0 &lt; 0 &gt; C and stirred for 20 minutes. Fuming nitric acid (6.2 mL, 144.1 mmol) was added and the reaction mixture was warmed to 22 &lt; 0 &gt; C and stirred for 3 hours. The reaction mixture was evaporated, MTBE (250 mL) was added and evaporated twice. The residue was dried in vacuo to constant weight and then dissolved in water (147 mL) and ethanol (885 mL) and degassed by bubbling nitrogen for 15 minutes. The solution was transferred to a pressure reactor and 10% Pd / C paste type 87L (6.6 g, 6.27 mmol) was added. The mixture was diluted with additional ethanol (700 mL) and pressurized with hydrogen at 80 psi for 16 h. The reaction mixture was filtered, then the second charge of the catalyst was added to 10% Pd / C paste type 87L (6.6 g, 6.27 mmol), the mixture was pressurized to 80 psi and stirred in a pressure reactor for 3 days. The reaction mixture was purged with nitrogen and filtered over diatomaceous earth. The filtrate was evaporated and the residue was partitioned between water (200 ml) and EtOAc (200 ml). The aqueous layer was separated, cooled to 5 &lt; 0 &gt; C and neutralized with 15% w / w ammonium hydroxide. The aqueous layer was extracted with EtOAc (3 x 150 mL). The organics were combined, dried over sodium sulfate, filtered and evaporated under reduced pressure to give the title compound as a pale brown solid (47.7 g, 99%, with residual EtOAc). ES / MS (m / e): 363 (M + 1).

Example A

3,4-d] [l, 3] thiazolo [3,4-d] pyrimidin- 7-yl] -4-fluoro-phenyl] -5-fluoro-pyridine-2-carboxamide

Dihydro-pyrrolo [3,4-d] [1,3] thiazepin-2-yl) 4-fluoro-phenyl] -5-fluoro-pyridine-2-carboxamide (293 mg, 497 μmol) and O- methylhydroxylamine hydrochloride (430 mg, 4.97 mmol) and pyridine (402 [mu] L, 4.97 mmol) was heated in ethanol (13 mL) at 70 <0> C for 2.5 h in a capped flask. DMSO (3 mL) was added and the mixture was heated at 70 &lt; 0 &gt; C overnight. Additional DMSO (10 mL) was added and heating was continued at 70 &lt; 0 &gt; C for 4 h. Additional O-methylhydroxylamine hydrochloride (208 mg, 2.48 mmol) and pyridine (201 [mu] L, 2.48 mmol) were added and the mixture was heated to 60 [deg.] C for 3 hours and the mixture was stirred at room temperature for 3 days . 3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] Yl] -4-fluoro-phenyl] -5-fluoro-pyridine-2-carboxamide (276 mg, 468 μmol), O-methylhydroxylamine hydrochloride 405 mg, 4.68 mmol) and pyridine (478 [mu] L, 4.68 mmol) were heated in a capped flask at 70 [deg.] C in ethanol (15 mL) and DMSO (4 mL) overnight. Additional DMSO (10 mL) was added and heating was continued at 70 &lt; 0 &gt; C for 4 h. Additional O-methylhydroxylamine hydrochloride (195 mg, 2.34 mmol) and pyridine (189 [mu] L, 2.34 mmol) were added and heated at 70 [deg.] C for 3 hours, then the mixture was stirred at room temperature for 3 days. The two reaction mixtures were combined and most of the solvent was removed under vacuum. The crude product was purified on a SCX column using 3: 1 DCM: methanol and then purified using 2: 1 DCM: 7 N ammonia in methanol. The crude product was further purified on silica gel with a 0.5% to 10% 20 min gradient of 7 N ammonia methanol in DCM gradient to give the title compound (451 mg, 96%). ES / MS (m / e): 486 (M + H).

Example 1

3,4-d] pyrimidin-2-yl) -4-tert-butoxycarbonylamino- [1,3] thiazin-7a (4H) -yl] phenyl} -5-fluoropyridine-2- carboxamide hydrochloride

To a solution of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- 2-yl) -4,4a, 5,7-tetrahydrofuran Yl] phenyl] -5-fluoro-pyridine-2-carboxamide (320 mg, 560 [mu] mol), O-methylhydroxylamine hydrochloride A mixture of chloride (485 mg, 5.60 mmol) and pyridine (453 [mu] L, 5.60 mmol) was heated at 65 [deg.] C for 5 h in a plugged vial. The reaction was cooled and the solvent was removed in vacuo. The crude product was purified on silica gel by gradient from 0.5% to 10% 30 minutes in a 7N ammonia gradient in methanol DCM to give N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidine Yl] phenyl] -5-fluoro-pyridine-2-carbaldehyde (219 mg, 84%). This material was dissolved in DCM (1 mL) and methanol (0.5 mL) and 1 M hydrogen chloride in diethyl ether (0.47 mL, 470 μmol) was added. The solvent was removed in vacuo to give the title compound (228 mg, 81%). ES / MS (m / e): 468 (M + H).

Example 2

3,4-d] pyrimidin-2-yl) -4-tert-butoxycarbonylamino- [1,3] thiazin-7a (4H) -yl] phenyl} -5-methoxypyrazine-2- carboxamide hydrochloride

To a solution of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- 2-yl) -4,4a, 5,7-tetrahydrofuran Yl] phenyl] -5-methoxy-pyrazine-2-carboxamide (479 mg, 819 μmol), O-methylhydroxylamine hydrochloride Chloride (709 mg, 8.19 mmol) and pyridine (663 [mu] L, 8.19 mmol) was heated in a capped flask at 50 [deg.] C overnight. DMSO (4 mL) was added and the mixture was heated at 70 &lt; 0 &gt; C for 4 hours to give a solution. The reaction was allowed to cool and most of the solvent was removed in vacuo. Water was added and the pH was adjusted to ~ 12 with 1 N sodium hydroxide. The mixture was extracted with EtOAc (5 x). The combined organic extracts were dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified on a silica gel gradient from 0.5% to 10% 30 minutes in a 7 N ammonia methanol gradient in DCM. The mixture was re-purified on a SCX column with 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol to remove residual DMSO. The mixture was finally purified on a silica gel with a 0.5% to 10% 20 min gradient of 7 N ammonia methanol in DCM to give N- [3- [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidine Yl] phenyl] -5-methoxy-pyrazine-2-carbaldehyde Lt; / RTI &gt; amide. This material was dissolved in DCM (1 mL) and methanol (0.5 mL) and 1 M hydrogen chloride in ether (0.66 mL, 660 μmol) was added. The solvent was removed in vacuo to give the title compound (329 mg, 78%). ES / MS (m / e): 481 (M + H).

Example 3

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7a-yl] -4-fluoro-phenyl] -5-fluoro-pyridine- 2- carboxamide hydrochloride

3,4-d] [1,3] thiazepin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4- d] ] Pyrazin-7-yl] -4-fluoro-phenyl] -5-fluoro-pyridine-2- carboxamide , 2.1 x 25 cm Eluent: chiral purification with 40% methanol in CO ₂ (0.2% isopropylamine, flow rate 70 mL / min, UV 225 nm) Chiral analysis of the first eluent: column: chiralcel OD- H (5 μm), 4.6 x 150 mm; eluent: CO of _240% methanol (0.2% isopropyl amine); flow rate 5 mL / min, enantiomers of UV 225 nm has R _t = 1.01 minutes, typically rich (> This material (free base, isomer 1) was dissolved in DCM (1 mL) and methanol (0.5 mL) and 1 M hydrogen chloride in diethyl ether ( The title compound (183 mg, 38%) was obtained by removal of the solvent under vacuum ES / MS (m / e): 486 (M + H).

Example 4

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide hydrochloride.

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- 4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide (0.350 g, 0.58 mmol, isomer 1) was dissolved in THF ), Followed by addition of methanol (4 mL) and ethanol (4 mL). O-methylhydroxylamine hydrochloride (495 mg, 5.81 mmol) and pyridine (470 [mu] L, 5.81 mmol) were added to the mixture and the reaction was warmed to 50 &lt; 0 &gt; C and stirred overnight. Silica gel (~ 10 g) was added to the reaction and the mixture was concentrated. The dried sample on silica gel was loaded onto an empty cartridge and purified by eluting with a 0-10% gradient of 7 N ammonia methanol in DCM. The product was again purified on a SCX column using 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol. The product was finally purified on a silica gel with 0% to 10% gradient of 7 N ammonia methanol in DCM to give the free base of the title compound. This material was dissolved in DCM (5 mL) and 1 M hydrogen chloride in diethyl ether (0.20 mL, 660 [mu] mol) was added. The solvent was removed in vacuo to give the title compound (71 mg, 23%). ES / MS (m / e): 498 (M + H).

Example 5

Crystalline form 2 &lt; RTI ID = 0.0 &gt; N- [3 - [(4aR, 7aS) -2-Amino- 6- (5-fluoropyrimidin- 2- yl) -4a, 5,6,7-tetrahydropyrrolo [ -d] [l, 3] thiazin-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide (hydrated).

Acetonitrile (500 mL) was added to dimethylformamide (19.2 mL, 248.9 mmol). Oxalyl chloride (39.3 g, 309.63 mmol) followed by 5-methoxypyrazine-2-carboxylic acid (46.0 g, 298.4 mmol) was added to the dimethylformamide solution. In a separate flask, (4aR, 7aS) -7a- (5-amino-2-fluorophenyl) -6- (5-fluoropyrimidin- (56.8 g, 156.75 mmol) was added to acetonitrile (500 mL) and the pH was adjusted to pH 7.5 with ammonium hydroxide (95 &lt; RTI ID = 0.0 &gt; mL ). &Lt; / RTI &gt; The mixture was then heated to 50-55 占 폚. The acid chloride solution was added dropwise, and the mixture was stirred for 3 hours. The pH was adjusted to 8-9 using ammonium hydroxide. The resulting precipitate was filtered, washed with water and dried to give the title compound (123 g). The solid was slurried in acetone (250 mL) for 1.5 hours and filtered. The wet cake was washed with acetone to give the title compound (110 g, 90.5% pure, by HPLC). THF (1 L) and activated charcoal (9 g) were added to the solid and the mixture was heated under reflux overnight. The mixture was filtered through diatomaceous earth and washed with THF (150 mL). The organic solution was concentrated to 10 volumes and heated to 60 &lt; 0 &gt; C. Water (430 mL) was added and the mixture was stirred at 60 &lt; 0 &gt; C for 8 hours. The mixture was cooled to room temperature and stirred for 10 hours. The resulting solid was filtered, washed with THF / water (7: 6) and dried to give the title compound (69 g, 88%). LC-MS: m / z = 499 (M + 1), purity: 98.3%.

¹ H NMR (400 MHz, DMSO-d ₆ )? Ppm 2.99-3.07 (m, 1H) 3.07-3.14 (m, 1H) 3.58-3.67 (D, J = 11.98, 8.72 Hz, 1H), 7.78 (s, 2H), 3.76-3.84 (m, 1H) - 7.89 (m, 2 H) 8.41 (s, 1H) 8.44 (s, 2H) 8.88 (s, 1H) 10.60 (s, 1H).

X-ray powder diffraction (XRD)

An XRD pattern of crystalline solid was obtained on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKa source (lambda = 1.54060 A) and a Vantec detector and operating at 35 kV and 50 mA. The sample was scanned at 4 to 40 degrees at 2 [theta] using a step size of 0.009 [deg.] At 2 [theta] and a scan rate of 0.5 sec / step and 0.6 mm divergence, 5.28 fixed scattering prevention slit, and 9.5 mm detector slit. The dry powder was packed onto a quartz sample holder and the smooth side was obtained using a glass slide. The crystal form diffraction pattern was collected at ambient temperature and relative humidity. In the case of any given crystal form, it is well known in the art of crystallography that the relative intensities of the diffraction peaks can vary due to the desired orientation resulting from factors such as crystal morphology and wetting. In the presence of the effect of the preferred orientation, the peak intensity is changed, but the characteristic peak position of the polymorph does not change. See, for example, The United States Pharmacopeia # 23, National Formulary # 18, pages 1843-1844, 1995. In addition, it is also well known in the crystallographic art that, in the case of any given crystal form, the angular peak position may vary slightly. For example, the peak position can be shifted due to variations in temperature or humidity at which the sample is analyzed, sample displacement, or the presence or absence of an internal standard. In this case, the peak position variability of &lt; RTI ID = 0.0 &gt; 0. 2 &lt; / RTI &gt; at 2 theta will take these potential variations into account without compromising the apparent identification of the crystalline form shown. Identification of the crystal form can be performed on the basis of a characteristic peak (in units of 2 &amp;thetas;), typically any unique combination of more pronounced peaks. The crystal form diffraction patterns collected at ambient temperature and relative humidity were adjusted based on the NIST 675 standard peak at 8.853 and 26.774 degrees 2-theta.

Crystalline form 2 &lt; RTI ID = 0.0 &gt; N- [3 - [(4aR, 7aS) -2-Amino- 6- (5-fluoropyrimidin- 2- yl) -4a, 5,6,7-tetrahydropyrrolo [ -d] [1,3] thiazin-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy- pyrazine- 2- carboxamide was treated with XRD using CuKa radiation (2-theta value) as shown in Table 2 below. Specifically, the pattern contained a peak at 11.8 ° with one or more peaks selected from the group consisting of 18.6 °, 19.3 °, and 26.7 ° with a tolerance for a diffraction angle of 0.2 °.

Table 2: X-ray powder diffraction peaks of crystalline Form 2 of Example 5

Example 6

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7a-yl] phenyl] -5-cyano-pyridine-2- carboxamide hydrochloride

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- yl] phenyl] -5-cyano-pyridine-2-carboxamide (360 mg, 0.59 mmol) was dissolved in ethanol (10 mL) and DCM . O-methylhydroxylamine hydrochloride (504 mg, 5.91 mmol) and pyridine (478 [mu] L, 5.91 mmol) were added and the reaction stirred at room temperature for the weekend (70 h). The reaction was warmed to 60 &lt; 0 &gt; C and stirred for 24 h. The reaction was concentrated to give the crude product, which was purified by silica gel chromatography using a 0-10% gradient of 7 N ammonia methanol in DCM to give the free base of the title compound. This material was dissolved in DCM (5 mL) and 1 M hydrogen chloride in diethyl ether (0.54 mL, 540 [mu] mol) was added. The solvent was removed in vacuo to give the title compound (240 mg, 75%). ES / MS (m / e): 475 (M + H).

Example 7

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] phenyl] -3,5-difluoro-pyridine-2- carboxamide hydrochloride

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- d] [1,3] thiazin-7a-yl] phenyl] -3,5-difluoro-pyridine-2- carboxamide (330 mg, 0.53 mmol) was dissolved in THF (10 mL). O-methylhydroxylamine hydrochloride (453 mg, 5.32 mmol) and pyridine (430 L, 5.91 mmol) were added and the reaction stirred at room temperature for the weekend (70 h). The reaction was warmed to 60 &lt; 0 &gt; C and stirred for 24 h. The mixture was concentrated on silica gel (~ 10 g) and purified by silica gel chromatography using a 0-10% gradient of 7 N ammonia methanol in DCM to give the free base of the title compound. This material was dissolved in DCM (5 mL) and 1 M hydrogen chloride in diethyl ether (0.49 mL, 490 μmol) was added. The solvent was removed in vacuo to give the title compound (159 mg, 54%). ES / MS (m / e): 486 (M + H).

Example 8

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -5-cyano-pyridine- 2- carboxamide hydrochloride

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- pyridin-2-carboxamide (133 mg, 0.22 mmol, isomer 1) was dissolved in THF (1 mL) ) And diluted with methanol (3 mL) and ethanol (3 mL). O-methylhydroxylamine hydrochloride (190 mg, 2.2 mmol) and pyridine (180 [mu] L, 2.2 mmol) were added. The reaction was warmed to 50 &lt; 0 &gt; C and stirred overnight. The mixture was concentrated on silica gel (~ 10 g) and purified by silica gel chromatography eluting with a 0-10% gradient of 7 N ammonia methanol in DCM. This material was re-purified on a SCX column using 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol. The mixture was finally purified on a silica gel with 0% to 10% gradient of 7 N ammonia methanol in DCM to give the free base of the title compound. This material was dissolved in DCM (5 mL) and 1 M hydrogen chloride in diethyl ether (0.27 mL, 270 [mu] mol) was added. The solvent was removed in vacuo to give the title compound (114 mg, 97%). ES / MS (m / e): 493 (M + H).

Example 9

3,4-d] pyrimidin-2-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7-yl] -4-fluoro-phenyl] -3,5-difluoro-pyridine- 2- carboxamide hydrochloride

Synthesis of N- [3 - [(4aR, 7aS) -2-benzamido-6- (5-fluoropyrimidin- pyridin-2-carboxamide (190 mg, 0.31 mmol, isomer 1) was dissolved in THF &lt; RTI ID = 0.0 & (1 mL) and diluted with methanol (3 mL) and ethanol (3 mL). O-methylhydroxylamine hydrochloride (267 mg, 3.1 mmol) and pyridine (253 L, 3.1 mmol) were added and the reaction was warmed to 50 &lt; 0 &gt; C and stirred overnight. The reaction was purified by SCX column using 3: 1 DCM: methanol followed by 2: 1 DCM: 7 N ammonia in methanol. This material was finally purified on a silica gel with 0% to 10% gradient of 7 N ammonia methanol in DCM to give the free base of the title compound. This material was dissolved in DCM (5 mL) and 1 M hydrogen chloride in diethyl ether (0.20 mL, 200 μmol) was added. The solvent was removed in vacuo to give the title compound (101 mg, 60%). ES / MS (m / e): 504 (M + H).

Example 10

3,4-d] pyrimidin-2-yl) -4a, 5,6,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-7a (4H) -yl] -4-fluoro-phenyl] -3,5-difluoro-pyridine- 2- carboxamide

Oxalyl chloride (10.5 ml, 136.4 mmol) was added to a solution of 3,5-difluoropicolinic acid (19.9 g, 125 mmol) in acetonitrile (617 mL) and dimethylformamide (10.5 mL) . After stirring for 30 min the solution was treated with a solution of (4aR, 7aS) -7a- (5-amino-2-fluoro-phenyl) -6- (5-fluoro 4-yl) -4,4a, 5,7-tetrahydropyrrolo [3,4-d] [1,3] thiazin-2-amine (41.1 g, 113 mmol) And preheated to 50 &lt; 0 &gt; C. The reaction was held at 50 &lt; 0 &gt; C for 1 hour. The reaction mixture was cooled to 22 &lt; 0 &gt; C and the solvent was evaporated. The aqueous solution was diluted with DCM (1 L) and the pH was adjusted to pH = 11 using 2 M sodium hydroxide. The organic layer was separated and the aqueous layer was washed with additional DCM (2 x 400 mL). The organic extracts were combined, dried over sodium sulfate, filtered and evaporated to dryness. The crude material was purified by silica gel chromatography using a gradient of ammoniated methanol 2 N / DCM from 0-10% DCM to give the title compound as an off-white solid (45 g, 78%). ES / MS (m / e): 504 (M + 1).

In vitro assay procedure:

For in vitro enzyme and cell assays, the test compound was prepared in DMSO to make a 10 mM stock solution. In vitro enzyme and whole cell assays were performed after serial dilution of the stock solution in DMSO to yield a 10-point dilution curve with final compound concentrations ranging from 10 mM to 0.05 nM in 96-well round-bottom plates.

In vitro protease inhibition assay:

Expression of human BACE1

Human BACE1 (accession number: AF190725) was cloned from total brain cDNA by RT-PCR. The nucleotide sequence corresponding to amino acid sequence # 1 to 460 was inserted into the cDNA encoding human IgG ₁ (Fc) polypeptide (see Vasser et al., Science, 286, 735-741 (1999)). This fusion protein of BACE1 (1-460) and human Fc designated huBACEl: Fc was constructed in pJB02 vector. Human BACE1 (1-460): Fc (huBACE1: Fc) was transiently expressed in HEK293 cells. 250 [mu] g cDNA of each construct was mixed with Fugene 6 and added to 1 liter HEK293 cells. After 4 days of transfection, the conditioned medium was harvested for purification.

huBACE1: purification of Fc

huBACE1: Fc was purified by protein A chromatography. The enzyme was stored at -80 &lt; 0 &gt; C in small aliquots.

BACE1 FRET Black

A series of dilutions of the test compound was prepared as described above. Compounds were further diluted 20x in KH ₂ PO ₄ buffer. 10 μL of each dilution was added to the reaction mixture (25 μL of 50 mM KH ₂ PO ₄ , pH 4.6, 1 mM TRITON® X-100, 1 mg / mL bovine serum albumin, and 15 μM FRET substrate) (Yang, et al., J. Neurochemistry, 91 (6) 1249-59 (2004)) of corresponding low protein binding black plates. The contents were mixed well on a plate shaker for 10 minutes. 15 μL of 200 pM human BACE1 (1-460): Fc (see Vasser, et al., Science, 286, 735-741 (1999)) in KH ₂ PO ₄ buffer was added to a plate containing the substrate and the test compound To initiate the reaction. After brief mixing on a plate shaker, the RFU of the mixture at time 0 was recorded at excitation wavelength 355 nm and emission wavelength 460 nm. The reaction plate was covered with aluminum foil and kept in a dark, humid oven at room temperature for 16-24 hours. At the end of the incubation the RFU was recorded with the same excitation and emission settings used at time zero. Time zero and the difference in RFU at the end of incubation represent the activity of BACEl under compound treatment. The RFU difference was plotted against the inhibitor concentration and the curve was substituted into the 4-parameter logistic equation to determine EC ₅₀ and IC ₅₀ values. (See Sinha, et al., Nature, 402, 537-540 (2000)).

The following exemplified compounds were tested essentially as described above and exhibited the following activity against BACEl:

Table 3

Mean ± SEM; SEM = standard error of mean

These data demonstrate that the compounds of Table 3 inhibit the purified recombinant BACE1 enzyme activity in vitro.

Whole cell assay to measure inhibition of beta-secretase activity

HEK293Swe whole cell assay

In a typical whole cell assay for measuring inhibition of beta-secretase activity, a human APP751 cDNA containing a spontaneous double mutation from Lys651Met652 to Asn651Leu652, often referred to as the Swedish mutation and shown to over-produce Abeta, Human embryonic kidney cell line HEK293p (ATCC Accession No. CRL-1573) (referred to as HEK293Swe) was used (Citron, et al., Nature, 360, 672-674 (1992)). In vitro A beta reduction assays have been described in the literature (Dovey, et al., Journal of Neurochemistry, 76, 173-181 (2001); Seubert, et al., Nature, 361, 260 Johnson-Wood, et al., Proc Natl Acad Sci USA, 94, 1550-1555 (1997)).

Cells (containing 200 μL of culture medium DMEM containing 3.5 x 10 ⁴ cells / well of HEK293Swe, 10% FBS) were incubated for 4 to 24 hours at 37 ° C. in the presence / absence of the desired concentration of inhibitor (diluted with DMSO) Lt; / RTI &gt; At the end of the incubation, conditioned media was analyzed for evidence of beta-secretase activity, for example, by assaying A beta peptide. Total A beta peptide (A beta 1-x) was measured by sandwich ELISA using monoclonal 266 as the capture antibody and biotinylated 3D6 as the reporting antibody. Alternatively, A beta 1-40 and A beta 1-42 peptides were used as capture antibodies in the case of A beta 1-40 and monoclonal 21F12 in the case of A beta 1-42 in the case of A beta 1-42 &Lt; / RTI &gt; by sandwich ELISA. A Beta 1-40 and A beta 1-42 ELISA both used biotinylated 3D6 as the reporting antibody. The concentration of A beta released into the conditioning medium after compound treatment corresponded to the activity of BACE1 under these conditions. The 10-point inhibition curve was plotted and substituted into the 4-parameter logistic equation to determine the EC ₅₀ and IC ₅₀ values for the A-beta-lowering effect. The following exemplified compounds were tested essentially as described above and exhibited the following activity for the A beta depot effect:

Table 4

These data demonstrate that the compounds in Table 4 strongly inhibit the natural A-beta production of whole cells.

PDAPP primary neuron assay

Confirmatory whole cell assays were also performed in primary neuron cultures generated from PDAPP transgenic embryo mice. Primary cortical neurons were prepared from embryonic day 16 PDAPP embryos and cultured in 96 well plates (15 x 10 ⁴ cells / well in DMEM / F12 (1: 1) plus 10% FBS). After 2 days in vitro, the culture medium was replaced with serum-free DMEM / F12 (1: 1) containing B27 supplement and 2 [mu] M (final) ara- C (Sigma, C1768) . On day 5 in vitro, neurons were incubated for 24 hours at 37 C in the presence / absence of the desired concentration of inhibitor (diluted in DMSO). At the end of the incubation, the conditioned medium was assayed, for example A beta peptide, for evidence of beta-secretase activity. Total A beta peptide (A beta 1-x) was measured by sandwich ELISA using monoclonal 266 as the capture antibody and biotinylated 3D6 as the reporting antibody. Alternatively, A beta 1-40 and A beta 1-42 peptides were used in the case of A beta 1-40 using monoclonal 21F12 as the capture antibody in the case of monoclonal 2G3 and A beta 1-42 as the capture antibody And measured by sandwich ELISA. A Beta 1-40 and A beta 1-42 ELISA both used biotinylated 3D6 as the reporting antibody. The concentration of A beta released into the conditioning medium after compound treatment corresponded to the activity of BACE1 under these conditions. The 10-point inhibition curve was plotted and substituted into the 4-parameter logistic equation to determine the EC ₅₀ and IC ₅₀ values for the A-beta-lowering effect. The following exemplified compounds were tested essentially as described above and exhibited the following activity for the A beta depot effect:

Table 5

Mean ± SEM; SEM = standard error of mean

These data demonstrate that the compounds of Table 5 strongly inhibit A-beta production of whole cells.

In vivo inhibition of beta-secretase

Several animal models including mice, guinea pigs, dogs, and monkeys can be used to screen for inhibition of in vivo beta-secretase activity following compound treatment. The animal used in the present invention may be a wild type, a transgenic, or a gene knockout animal. For example, a PDAPP mouse model, and other non-transgenic or gene knockout animals prepared as described in [Games et al., Nature 373, 523-527 (1995)], Beta &lt; / RTI &gt; and sAPP beta production. Generally, a compound formulated in a vehicle such as corn oil, cyclodextran, phosphate buffer, PHARMASOLVE®, or other suitable vehicle is administered to a PDAPP mouse, a gene knockout mouse or a non-transgenic animal at 2-12 months of age, . After 1 to 24 hours of compound administration, the animals were sacrificed and cerebrospinal fluid and plasma as well as brain were removed for analysis of A beta, C99 and sAPP fragments. (See May, et al., Journal of Neuroscience, 31, 16507-16516 (2011)).

For standard in vivo pharmacokinetic studies, animals were dosed with various concentrations of compound and compared to vehicle-treated controls administered at the same time. For some time course studies, brain tissue, plasma or cerebrospinal fluid was obtained from selected animals starting at time 0, establishing a baseline. Compound or appropriate vehicle was administered to another group and sacrificed at various time points after administration. Brain tissue, plasma or cerebrospinal fluid was obtained from selected animals and analyzed by, for example, specific sandwich ELISA assays for the presence of APP cleavage products including A beta peptide, sAPP beta and other APP fragments. At the end of the test period, animals were sacrificed and brain tissue, plasma or cerebrospinal fluid was analyzed for the presence of A beta peptide, C99 and sAPP beta, as appropriate. The brain tissue of the APP transgenic animals was also analyzed for the amount of beta-amyloid plaques after compound treatment. As used herein, "A beta 1-x peptide" refers to the sum of the A beta species beginning with residue 1 and terminating at the C-terminus of residue 28. This detects most A beta species and is often referred to as "total A beta. &Quot;

Animal (PDAPP or other APP transgenic or non-transgenic mice) receiving the inhibitory compound showed a decrease in A beta or sAPP beta in brain tissue, plasma or cerebrospinal fluid, The reduction of beta amyloid plaques in the tissues can be demonstrated. Three-hour post-dosing of the 1, 3 or 10 mg / kg oral dose of the compound of Example 1 to the young female PDAPP mice resulted in an increase in the level of A beta 1-x peptide compared to the vehicle- 34%, 48% and 53%, and approximately 43%, 59% and 66% in the cerebral cortex.

Three hours after the administration of 1 or 3 mg / kg oral dose of the compound of Example 3, the level of A beta 1-x peptide was approximately 38% and 50%, respectively, in the brain hippocampus compared to the vehicle- In the cortex by approximately 34% and 53%.

In Example 4, three hours after administration of 0.3, 1 or 3 mg / kg oral doses of compound, the A beta 1-x peptide levels were approximately 31%, 39% And 61%, and approximately 28%, 42% and 64% in the cerebral cortex.

Taking into account the activity of Examples 1, 3 and 4 on in vitro BACE enzymes, this A-beta depot effect is consistent with in vivo BACE inhibition and further demonstrates CNS infiltration in Examples 1, 3, and 4 .

These studies suggest that the compounds of Examples 1-9 inhibit BACE and thus are useful for decreasing A beta levels.

Combination Studies

BACE Inhibitor Feed Pilot Experiment

Pilot pharmacokinetic and pharmacodynamic studies have shown that a BACE inhibitor, such as a feed containing a compound of formula I or a pharmaceutically acceptable salt thereof, to define a dose that provides minimal or significant plasma and brain A beta reduction by BACE inhibition alone Diet was performed by feeding PDAPP mice. Young PDAPP mice received diets containing feed diets containing a "quasi-bid" equivalent dose of BACE inhibitors of 3 mg / kg, 10 mg / kg, 30 mg / kg or 100 mg / kg for 14 days. ~ 0.05, 0.15, 0.5 or 1.5 mg of BACE inhibitor per gram of authentic rodent diet # 8728CM (Harlan labs) was mixed in a Sorvall mixer for 10 minutes prior to pelleting, followed by 15 minutes Lt; / RTI &gt; in a Hobart mixer. Thirty-two young female PDAPP mice were randomized according to the parental cell line into four groups of eight animals consisting of a vehicle-treated group and a three-dose BACE inhibitor. Mice were allowed to have unrestricted access to food for 14 days and subsequently sacrificed. Mice were anesthetized with CO ₂ and blood was collected by cardiac puncture with EDTA-coated microcentrifuge tubes and stored on ice. Subsequently, the plasma was collected by centrifugation of the blood sample at 14,000 rpm for 4 minutes at room temperature, transferred to untreated microcentrifuge tubes, frozen on dry ice, and stored at -80 DEG C until analysis. Mice were sacrificed by monocots, the brain was rapidly micro-dissected in half, frozen on dry ice, and stored at-80 C until analysis (one half for A beta analysis and the other half for compound exposure measurement ). For analysis of parenchymal A beta, brain samples were homogenized in 5.5 M guanidine-HCl buffer (0.5 mL per half-brain) with a tissue titer (Model 985-370) at a rate of 5 for about 1 minute. Homogenized brain samples were allowed to thaw at room temperature overnight.

For A-beta ELISA analysis, the extracts were collected and resuspended in 1x PBS with 0.25% casein, 0.05% Tween 20, 0.1% thimerosal, pH 7.4 with protease inhibitor cocktail (Sigma P9340 at 0.01 mg / ) At least 1:10 and centrifuged at 14000 rpm for 10 minutes. For analysis of plasma A beta, samples were diluted 1: 2 with sample buffer (PBS; 0.05% Triton X-405; 0.04% thimerosal, 0.6% BSA) prior to analysis by ELISA. A human plasma A beta _{1 -x} was determined by sandwich ELISA using each m266.2 (wherein -A beta _13-28) and biotinylated 3D6 (wherein -A beta 1-5) as capture antibody and a reporter. Unknowns were interpolated from an 8-point standard curve (Soft Max Pro v. 5.0.1, Molecular Dynamics) using 4-parameter feet of the reference curve and then adjusted for dilution Double analysis and determination of pg / mL. The parenchymal A beta was determined by sandwich ELISA as described above and the value was normalized to the protein level (doubly determined by the Bradford Coomassie Plus Protein method) and expressed as pg / mg protein .

To determine the tissue and plasma levels of the BACE inhibitor, the following procedure was used: working solution was prepared by continuously diluting the 0.1 mg / mL stock solution of BACE inhibitor with methanol / water (1: 1, v / v) This was then used to fortify control plasma and brain homogenates to yield analyte concentrations of 1, 5, 10, 20, 50, 100, 500, 1000, 2000, 4000 and 5000 ng / mL. Prior to analysis, brain samples were homogenized using a sonicator disrupter in a 3-fold volume of methanol / water (1: 4, v / v). An aliquot of each study sample, an appropriate calibration standard, and a control matrix sample was transferred to a 96-well plate and then mixed with acetonitrile containing an internal standard. After mixing, the sample was centrifuged to pellet the precipitated protein. An aliquot of the resulting supernatant was then transferred to a clean 96-well plate, diluted with methanol / water (1: 1, v / v), and 10 microliters aliquots were analyzed by LC-MS / MS. The analyte concentration was calculated using the response to the concentration relationship determined by multiple regression of the calibration curve samples.

In vivo combination study

Anti-N3pGlu A beta monoclonal antibody such as anti-N3pGlu-A beta monoclonal antibody VII (see Table 1, mE8c-IgG2a; as described in U.S. Patent No. 8,679,498 B2; USSN 13 / 810,895) To evaluate the combined plaque-lowering regimens of the compounds of formula (I) or their pharmaceutically acceptable salts, large cohorts of PDAPP mice were first aged to 16-18 months of age. Aged PDAPP mice were randomized into 5 treatment arms based on sex, parental cell line, and age. There were 20 to 30 aged PDAPP mice per treatment arm. Group 1 was sacrificed at time zero in study initiation to determine baseline levels of pathology prior to healing (described below). The remaining four groups were then treated as follows: control animals received weekly injections of group 2, placebo feed diets and 12.5 mg / kg of control isotype IgG2a; Group -3, animals receiving weekly injections of anti-N3pGlu-A beta monoclonal antibody at 12.5 mg / kg; Group -4, an animal previously defined in the pilot supply study, but typically receiving a BACE inhibitor feed diet of ~ 3 to 30 mg / kg / day; Animals receiving a weekly injection of group 5, BACE inhibitor feed diets (~ 3 to 30 mg / kg / day) and 12.5 mg / kg of anti-N3pGlu-A beta monoclonal antibody. The anti-N3pGlu-A beta monoclonal antibody was diluted from sterile stock solution of antibody in PBS buffer and administered to animals by intraperitoneal injection. BACE inhibitors were mixed with a loose feed diet (~ 0.15 to 1.5 mg compound per gram of feed according to the desired dose) and compressed with feed pellets. The animal weights were recorded weekly for the first month of study initiation and subsequent treatment, weekly for the duration of the study. Food intake was also monitored at regular intervals throughout the course of the study. The animals received research treatments for a total of four months. Animals maintained their respective diets until autopsy occurred after 1 week of final antibody injection. At the time of autopsy, the animals were anesthetized and blood was obtained by cardiac puncture using a 1 ml syringe with EDTA pre-washed. Blood samples were collected on ice and plasma was isolated by standard centrifugation. Subsequently, the animals were perfused with cold heparinized saline, the brain was removed, and dissected into right and left hemispheres. One brain hemisphere was rapidly frozen and stored for histological analysis. The remaining brain hemispheres were dissected with tissue sections composed of hippocampus, cortex, cerebellum and midbrain, and subsequently frozen on dry ice. Plasma and tissue samples were stored at -80 &lt; 0 &gt; C until analysis.

Pharmacokinetic evaluation

Plasma pharmacokinetics were determined on plasma samples obtained at the time of autopsy. Plasma antibody levels were determined in an antigen binding ELISA assay where the plates were coated with antigen (A beta _p3-42 ) and incubated with anti-N3pGlu monoclonal antibody in a subsequently diluted plasma sample or black buffer (PBS + murine plasma control) Incubated with a reference standard consisting of serial dilutions of the anti-mAb. After washing the plates, bound murine antibodies were detected by color expression with anti-murine-HRP conjugated antibodies followed by TMB. The following method was used to determine the tissue (midbrain) and plasma levels of the BACE inhibitor: 0.1 mg / mL of the BACE inhibitor stock solution was serially diluted with methanol / water (1: 1, v / v) , Which was then used to fortify control plasma and brain homogenates to yield analyte concentrations of 1, 5, 10, 20, 50, 100, 500, 1000, 2000, 4000 and 5000 ng / mL. Prior to analysis, brain samples were homogenized using a sonicator disrupter in a 3-fold volume of methanol / water (1: 4, v / v). An aliquot of each study sample, an appropriate calibration standard, and a control matrix sample was transferred to a 96-well plate and then mixed with acetonitrile containing an internal standard. After mixing, the sample was centrifuged to pellet the precipitated protein. An aliquot of the resulting supernatant was then transferred to a clean 96-well plate, diluted with methanol / water (1: 1, v / v), and 10 microliters aliquots were analyzed by LC-MS / MS. The analyte concentration was calculated using the response to the concentration relationship determined by multiple regression of the calibration curve samples.

Pharmacodynamic evaluation

The real A beta concentration was determined in a guanidine solubilized tissue homogenate by sandwich ELISA. Tissue extraction was performed with bead beater technology in which the frozen tissue was incubated with 5.5 M guanidine / 50 mM tris / 0.5X protease inhibitor cocktail at pH 8.0 in a 2 ml deep well dish containing 1 ml of siliconized glass beads 1 ml (sealed plates were shaken for two-thirds of each time). The resulting tissue lysates were analyzed by sandwich ELISA for A beta _{1 -40} and A beta _{1 -42} : bead beater samples were diluted 1: 10 in 2% BSA / PBS-T and stained on a sample plate (Millipore Millipore). Samples, blank, standard, quality control samples were further diluted in 0.5% M guanidine / 5 mM tris in 2% BSA / PBST prior to loading on the sample plates. Reference standards were diluted in the sample diluent. Plates coated with 15 μg / ml of capture antibody 21F12 (anti-A beta ₄₂ ) or 2G3 (anti-A beta ₄₀ ) were incubated with the samples and detection was performed by biotinylation with 2% BSA / PBS- 3D6 (anti-A beta _{1 -x} ) followed by 1:20 K diluted neutavidin-HRP (Pierce) and TMB (Pierce) in 2% BSA / PBS-T. A beta levels were interpolated from the standard curve and the final tissue concentration was calculated as nanograms of A beta per milligram of tissue wet weight. The percent area of hippocampus and cortex occupied by the deposited A beta was determined histologically. Cryopreserved continuous tubular sections (7-10 μm thick) were incubated with 10 μg / ml biotinylated 3D6 (anti-A beta _{1 -x} ) or negative control murine IgG (biotinylation). A second HRP reagent specific for biotin was used and the deposited A beta was visualized with DAB-Plus (DAKO). Immunoreactive A beta deposits were quantitated to the defined area of interest in the hippocampus or cortex by analyzing images captured with Image Pro plus software (Media Cybernetics).

These studies suggest that a combination therapy of an anti-N3pGlu-A beta monoclonal antibody and a BACE inhibitor, such as a compound of formula I, or a pharmaceutically acceptable salt thereof, may produce enhanced A beta reduction over individual monotherapy can do.

                         SEQUENCE LISTING <110> Eli Lilly and Company        MAY, Patrick        MERGOTT, Dustin   <120> COMBINATION THERAPY <130> P20348 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Ala Val Ser Lys Leu Asp Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Val Gln Gly Thr His Tyr Pro Phe Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Gly Tyr Asp Phe Thr Arg Tyr Tyr Ile Asn 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Gly Tyr Thr Phe Thr Arg Tyr Tyr Ile Asn 1 5 10 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Trp Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Glu Gly Ile Thr Val Tyr 1 5 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Glu Gly Thr Thr Val Tyr 1 5 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Arg Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Ala Val Ser Lys Leu Asp Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly                 85 90 95 Thr His Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 10 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Arg Tyr             20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Thr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr             100 105 110 Val Ser Ser         115 <210> 11 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr             20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Thr Thr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr             100 105 110 Val Ser Ser         115 <210> 12 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Arg Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Ala Val Ser Lys Leu Asp Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly                 85 90 95 Thr His Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu         115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe     130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu             180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser         195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 13 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Arg Tyr             20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Thr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr             100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro         115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val     130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly                 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly             180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys         195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys     210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu                 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys             260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys         275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu     290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser             340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys         355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln     370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln                 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly         435 440 <210> 14 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr             20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Asn Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Thr Thr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr             100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro         115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val     130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly                 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly             180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys         195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys     210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu                 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys             260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys         275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu     290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser             340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys         355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln     370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln                 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly         435 440

Claims (21)

A pharmaceutical composition for treating Alzheimer's disease in a patient, comprising an effective amount of a compound of the formula: or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an anti-N3pGlu A beta monoclonal antibody.

The pharmaceutical composition of claim 1, wherein the anti-N3pGlu A beta monoclonal antibody is B12L or R17L. 3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-N3pGlu A beta monoclonal antibody is B12L. 3. The compound of claim 1 or 2, wherein the compound is N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidin- -Tetrahydro- pyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5- methoxy-pyrazine- Composition. 3. A compound according to claim 1 or 2, wherein the compound is selected from the group consisting of crystalline form 2 N- [3 - [(4aR, 7aS) -2- amino- 6- (5- fluoropyrimidin- 6,7-tetrahydropyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5- methoxy-pyrazine- Amide. 6. The compound according to claim 5, wherein the crystalline form 2 is selected from the group consisting of N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidin- Pyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy- Wherein a substantial peak in the X-ray diffraction spectrum is characterized by a diffraction angle 2-theta of 11.8 DEG with one or more peaks selected from the group consisting of 18.6 DEG, 19.3 DEG and 26.7 DEG in terms of tolerance to . 3. The pharmaceutical composition according to claim 1 or 2, wherein the compound and the anti-N3pGlu A beta monoclonal antibody are administered simultaneously. 3. The pharmaceutical composition according to claim 1 or 2, wherein the compound is administered prior to the administration of the anti-N3pGlu A beta monoclonal antibody. 3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-N3pGluA beta monoclonal antibody is administered prior to the administration of the compound. A pharmaceutical composition comprising a compound of the formula: or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutical composition comprising a anti-N3pGlu A beta monoclonal antibody and one or more pharmaceutically acceptable carriers, diluents or excipients, in combination with one or more pharmaceutically acceptable carriers , &Lt; / RTI &gt; a diluent or an excipient, for the treatment of Alzheimer's disease in a patient.

11. The pharmaceutical composition according to claim 10, wherein the anti-N3pGluA beta monoclonal antibody is B12L or R17L. 12. The pharmaceutical composition according to claim 10 or 11, wherein the anti-N3pGlu A beta monoclonal antibody is B12L. 12. A compound according to claim 10 or 11 wherein the compound is N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidin- -Tetrahydro- pyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5- methoxy-pyrazine- Composition. 12. The compound according to claim 10 or 11, wherein the compound is selected from the group consisting of crystalline form 2 N- [3 - [(4aR, 7aS) -2-amino-6- (5- 6,7-tetrahydropyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5- methoxy-pyrazine- Amide. 15. The compound according to claim 14, wherein the crystalline form 2 is selected from the group consisting of N- [3 - [(4aR, 7aS) -2-amino-6- (5-fluoropyrimidin- Pyrrolo [3,4-d] [1,3] thiazine-7a (4H) -yl] -4-fluoro-phenyl] -5-methoxy- Wherein a substantial peak in the X-ray diffraction spectrum is characterized by a diffraction angle 2-theta of 11.8 DEG with one or more peaks selected from the group consisting of 18.6 DEG, 19.3 DEG and 26.7 DEG in terms of tolerance to . delete delete delete delete delete delete